DEFINITIVE ANNUAL REPORT FLOW CYTOMETRY: LYMPHOCYTE SUBSET ANALYSIS CD34+ STEM CELL ENUMERATION 2015

Size: px

Start display at page:

Download "DEFINITIVE ANNUAL REPORT FLOW CYTOMETRY: LYMPHOCYTE SUBSET ANALYSIS CD34+ STEM CELL ENUMERATION 2015"

Derrick Norris
5 years ago
Views:

EXPERTISE, SERVICE PROVISION AND CUSTOMER RELATIONS QUALITY OF MEDICAL LABORATORIES CLINICAL BIOLOGY COMMISSION COMMITTEE OF EXPERTS EXTERNAL QUALITY ASSESSMENT IN CLINICAL BIOLOGY DEFINITIVE ANNUAL

1 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 2015 IPH/Flow cytometry/56 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. De Schouwer P. TEL: 03/ FAX: 03/ Dr. Gothot A. TEL: 04/ FAX: 04/ Dr. Hougardy N. TEL: 063/ FAX: 063/ Dr. Kestens L. TEL: 03/ FAX: 03/ Dr. Kornreich A. TEL: 071/ FAX: 071/ Dr. Mullier F. TEL: 081/ FAX: 081/ Dr. Pradier O. TEL: 02/ FAX: 02/ Dr. Van Bockstaele D. TEL: 015/ FAX: 015/ Dr. Vander Meeren S. TEL: 02/ FAX: 02/ Dr. Willemse J. TEL: 014/ FAX: 014/ Committee of experts: 26/04/2016 Authorisation to release report: by Marjan Van Blerk, scheme coordinator, on 13/06/2016 All the reports are also available on our webpage: FORM 43/125/E v6. 2/28

3 TABLE OF CONTENTS 1. LYMPHOCYTE SUBSET ANALYSIS Surveys Methodology of the Belgian clinical laboratories Results Pz evaluation CD34+ STEM CELL ENUMERATION Surveys Methodology of the Belgian clinical laboratories Results Pz evaluation 28 FORM 43/125/E v6. 3/28

4 1.1. Surveys 1. LYMPHOCYTE SUBSET ANALYSIS A triannual external quality assessment scheme for lymphocyte immunophenotyping is operational in Belgium since 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 of the send-out of the control material (day 0). In 2015, surveys were conducted in February (FC 13300, FC 13301, FC 13302), May (FC 13480, FC 13481, FC 13482) and October (FC 13782, FC 13783, FC 13784). 1 Canadian, 1 Latvian, 2 clinical trial laboratories and 46 Belgian clinical laboratories participated in these surveys Methodology of the Belgian clinical laboratories survey 2015/3 (n=45) Seven laboratories (15.6%) used a single platform approach for determining the absolute lymphocyte subset counts. Of these laboratories, 4 used Flow-Count beads (Beckman-Coulter) and 3 Trucount technology (BD Biosciences). Following tables provide an overview of the haematology analysers and flow cytometers used: Haematology analyser Number of participants Sysmex XE 2100/XE Sysmex XN 3000/ XN Siemens Advia Beckman Coulter UniCel DxH Abbott Cell-Dyn Sapphire 3 Sysmex XT 2000i/XT 4000i 2 Sysmex XS 1000i 2 Abbott Cell-Dyn Ruby 1 Not mentioned 3 FORM 43/125/E v6. 4/28

5 Flow cytometer Number of participants BD Biosciences FACSCanto II 20 Beckman Coulter Navios 10 Beckman Coulter Cytomics FC BD Biosciences FACSCalibur 4 Abbott Sapphire 1 BD Biosciences FACSCanto 1 BD Biosciences FACSCan 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, 77.5% also make use of commercial control material. CD3+, CD4+, CD8+, CD19+, and NK cells (44 participants) All laboratories applied the whole blood lysis technique. More than half of the laboratories (55.0%) used a lyse no wash procedure. The following table summarises the lysing reagents used: Lysing reagent Number of laboratories BD Biosciences FACS Lyse 22 Beckman-Coulter VersaLyse 5 Ammonium chloride (NH 4 Cl) 4 Beckman-Coulter Optilyse C 3 BD Biosciences Pharm Lyse 3 Beckman-Coulter Immunoprep reagent system 2 Not mentioned 5 Most laboratories used 4- or 6-colour combinations (n=41). Number of participants CD3 + CD4 + CD8 + CD19 + NK 3 colours colours colours colours colours colours FORM 43/125/E v6. 5/28

6 Belgian consensus recommendations have been issued for the performance of many flow cytometric-based tests (Acta Clinica Belgica 1999; 54:88-98). All but 1 laboratory 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 14 participants (CD56 (n=13) or CD16 (n=1) alone) used the combination of CD16 and CD56 to identify NK cells (n=41). All laboratories that have mentioned their gating technique (n=39) 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 12 Lymphosum % CD45 positive cells 1 + lymphosum 2 9 Lymphosum 2 + CD3 consistency check % CD45 positive cells 1 2 Not mentioned 4 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: 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: 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 (35 participants) All laboratories performed 2 (42.9%) or more (57.1%) washing steps. More than two thirds of the participants (73.5%) 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 v6. 6/28

7 1.3. Results 57.8% (2015/2) to 100% (2015/1) of the Belgian clinical laboratories mentioning the day of receipt got the blood samples on day 1 and 0% (2015/1) to 42.2% (2015/2) received the blood samples on day 2 (day 0: send-out of blood samples). 53.3% (2015/2) to 84.8% (2015/1) of the Belgian clinical laboratories indicating the day of sample testing performed the analyses on day 1 and 15.2% (2015/1) to 40.0% (2015/2) 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: (toolkit). All but 2 participants returned their results this way. FORM 43/125/E v6. 7/28

8 The following tables show the medians and coefficients of variation obtained for the different parameters on the samples sent in 2015: WBC 10 9 /L FC FC FC FC FC FC FC FC FC Lymphocytes % Haematology analyser FC FC FC FC FC FC FC FC FC Lymphocytes % Flow cytometer FC FC FC FC FC FC FC FC FC FORM 43/125/E v6. 8/28

9 CD3 % FC FC FC FC FC FC FC FC FC CD /L FC FC FC FC FC FC FC FC FC CD4 % FC FC FC FC FC FC FC FC FC FORM 43/125/E v6. 9/28

10 CD /L FC FC FC FC FC FC FC FC FC CD8 % FC FC FC FC FC FC FC FC FC CD /L FC FC FC FC FC FC FC FC FC FORM 43/125/E v6. 10/28

11 CD19 % FC FC FC FC FC FC FC FC FC CD /L FC FC FC FC FC FC FC FC FC NK % FC FC FC FC FC FC FC FC FC FORM 43/125/E v6. 11/28

12 NK 10 9 /L FC FC FC FC FC FC FC FC FC κ % B lymphocytes FC FC FC FC FC FC FC FC FC λ % B lymphocytes FC FC FC FC FC FC FC FC FC FORM 43/125/E v6. 12/28

13 κ/λ ratio FC FC FC FC FC FC FC FC FC κ+λ % B lymphocytes FC FC FC FC FC FC FC FC FC Lymphosum % FC FC FC FC FC FC FC FC FC The CVs for the WBC count ranged between 3.2 and 5.5%. The CVs for the % lymphocytes ranged from 3.2 to 5.5% for the % lymphocytes obtained with haematology analysers and ranged from 5.1 to 10.3% for the % lymphocytes obtained with flow cytometers. For the different lymphocyte subsets, the average between-laboratory variability was 2.8, 3.5, 4.9, 12.0, and 12.6% for the % of CD3+, CD4+, CD8+, CD19+, and NK cells, respectively. The average CVs of the absolute values were higher and amounted to 7.1, 7.9, 8.6, 14.8, and 16.0% for CD3+, CD4+, CD8+, CD19+, and NK cells, respectively. The overall CVs were larger for small subsets. FORM 43/125/E v6. 13/28

14 For the percentages of κ and λ chain expressing B cells and the κ/λ ratio, the average CVs were 4.3, 5.7, and 8.2%, 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 (%) Year CD3 CV (%) Year FORM 43/125/E v6. 14/28

15 CD4 % CV (%) Year CD4 CV (%) Year FORM 43/125/E v6. 15/28

16 CD8 % CV (%) Year CD8 CV (%) Year FORM 43/125/E v6. 16/28

17 CD19 % CV (%) Year CD19 CV (%) Year FORM 43/125/E v6. 17/28

18 NK % CV (%) Year NK CV (%) Year FORM 43/125/E v6. 18/28

19 kappa CV (%) Year lambda CV (%) Year FORM 43/125/E v6. 19/28

20 kappa/lambda CV (%) Year FORM 43/125/E v6. 20/28

21 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 2015: Pz (n=46) Cumulative proportion of laboratories (%) Proportion of z-scores <-3 or >3 FORM 43/125/E v6. 21/28

22 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 Lymphocytes % HA Lymphocytes % FC CD3 % CD /L CD4 % CD /L CD8 % CD /L CD19 % CD /L NK cells % NK cells 10 9 /L κ % B lymphocytes λ % B lymphocytes κ/λ ratio κ+λ % B lymphocytes Lymphosum 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 CD /L CD /L CD /L CD /L NK cells 10 9 /L Parameter Number of evaluated results % results >3 SD Lyse and wash Lyse no wash Lyse and wash Lyse no wash CD3 % CD /L CD4 % CD /L CD8 % CD /L CD19 % CD /L NK cells % NK cells 10 9 /L Lymphosum For CD19 and NK, laboratories using a lyse no wash procedure obtained significantly less frequently results beyond 3 SD (Fisher s exact test). FORM 43/125/E v6. 22/28

23 Parameter Number of evaluated results % results >3 SD Monoclonal Polyclonal Monoclonal Polyclonal anti-κ/anti-λ anti-κ/anti-λ anti-κ/anti-λ anti-κ/anti-λ reagent reagent reagent reagent κ % B lymphocytes λ % B lymphocytes κ/λ ratio κ+λ % B lymphocytes For the κ and λ % B lymphocytes and 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 since 2011: 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 ± ± ± ± ± The maximum of evaluated results per laboratory was 162. This table shows a.o. that Belgian laboratories reported an average of 5.4% results beyond 3 SD in 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 v6. 23/28

24 2.1. Surveys 2. CD34+ STEM CELL ENUMERATION A triannual external quality assessment scheme for CD34+ stem cell enumeration is operational in Belgium since 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 acquisition, 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 2015, surveys were conducted in February (FC 13303, FC 13304), May (FC 13483, FC 13484) and October (FC 13785, FC 13786). Twenty-five Belgian clinical laboratories, 1 clinical trial laboratory, and 1 commercial cord blood bank participated in these surveys. The samples were sent by Taxipost 24h and the laboratories were informed by of the send-out of the control material (day 0). The laboratories were able to submit their results over the internet using the url: (toolkit). All but 2 participants returned their results this way Methodology of the Belgian clinical laboratories survey 2015/3 (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 14 Beckman-Coulter Navios 6 Beckman-Coulter Cytomics FC BD Biosciences FACSCanto 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, 72.0% of them also make use of commercial control material. FORM 43/125/E v6. 24/28

25 Sample preparation Thirteen participants used a sample volume of 50 µl, 11 participants a sample volume of 100 µl and 1 participant a sample volume of 30 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 2 BD Biosciences Ammonium chloride lysing solution 2 Beckman-Coulter Ammonium chloride 2 Cytognos Quicklysis 1 Qiagen EL-buffer 1 Monoclonal antibodies All but 2 laboratories (PC5.5/PE-Cy5.5, APC) used a phycoerythrin (PE)-conjugated CD34 monoclonal antibody. All but 6 participants (Horizon V500 (n=3), APC-H7, 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). Gating strategy With 3 exceptions (Beckman-Coulter Stem-Kit (1), BD Biosciences ProCount Kit (1), 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 v6. 25/28

26 2.3. Results 96.0% (2015/3) to 100% (2015/1 and 2015/2) of the participants mentioning the day of receipt got the blood samples on day 1 and 0% (2015/1 and 2015/2) to 4.0% (2015/3) received the blood samples on day 2 (day 0: send-out of blood samples). 84.0% (2015/3) to 92.0% (2015/1) of the participants indicating the day of sample testing performed the analyses on day 1 and 8.0% (survey 2015/1) to 12.5% (survey 2015/2 and 2015/3) 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 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 2015: Sample Median CV N Median CV N % CD34+ cells within total WBC % CD34+ cells/µl % FC FC FC FC FC FC The median CD34+ cell count ranged between 7.0 and 39.6 CD34+ cells/µl. The overall CV varied from 15.9 to 24.0%. FORM 43/125/E v6. 26/28

27 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 (%) Year FORM 43/125/E v6. 27/28

28 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 (2013: n=12, 2014: n=12, 2015: n=12), the P Z evaluation was based on the results obtained over 3 years. The next graph depicts the distribution of the P Z values of the participating laboratories for the period : Pz (n=28) Cumulative proportion of laboratories (%) Proportion of z-scores <-3 or >3 The maximum of evaluated results per laboratory was 36. Thirteen Belgian clinical laboratories (48.2%) reported no results beyond 3 SD in 2013, 2014, and Five laboratories (18.5%) reported 1 result, 2 laboratories (7.4%) 2 results, 2 other laboratories (7.4%) 3 results, 4 laboratories (14.8%) 4 results and 1 laboratory 5 results beyond 3 SD in 2013, 2014, and In 2015, 3 Belgian laboratories no longer participated in the scheme. Of the other participants, the vast majority (16 (64.0%)) reported no result beyond 3 SD. Two laboratories (8.0%) reported 1 result, 5 laboratories (20.0%) 2 results and 2 laboratories (8.0%) 3 results beyond 3 SD. 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 3 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. END Scientific Institute of Public Health, Brussels This report may not be reproduced, published or distributed without the consent of the WIV-ISP. FORM 43/125/E v6. 28/28

DEFINITIVE ANNUAL REPORT FLOW CYTOMETRY: LYMPHOCYTE SUBSET ANALYSIS CD34+ STEM CELL ENUMERATION 2014

DEFINITIVE ANNUAL REPORT FLOW CYTOMETRY: LYMPHOCYTE SUBSET ANALYSIS CD34+ STEM CELL ENUMERATION 2014 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