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National evaluation of IMAGN 2000 for quality monitoring of leucodepleted red cell and platelet concentrates: comparison with flow and Nageotte
Transfusion Science 22 (2000) 77±79
www.elsevier.com/locate/transci
National evaluation of IMAGN 2000 for quality monitoring of leucodepleted red cell and platelet concentrates: comparison with ¯ow and Nageotte J. Seghatchian a,*, P. Krailadsiri a, B. Chandegra a, M. Beard a, N. Beckman b, L. Bissett b, B. Morris c, W. Booker c a
National Blood Transfusion Service, Pro®cency Testing Laboratory, London & South East, UK b National Blood Transfusion Service, Birmingham, UK c National Blood Transfusion Service, Sheeld, UK
1. Introduction The urgent need for the implementation of universal leucodepletion of blood components in UK has led to rapid national evaluation of various types of commercially available ®ltration technologies for eective removal of leucocyte in blood components. Prerequisite to achieve this objective is the establishment of a precise and standardised methodology, in various production sites, for sampling and counting the residual leucocytes. To be of relevance the measurement should be carried out on ``real life'' samples, without additional processing, to re¯ect the true nature of the product for clinical use. The method of choice for leucocyte counting must be highly sensitive to small changes in the ``decision making point'', i.e., WBC <1 106 and <5 106 /unit both to allow improvements in leuco®ltration technology, and to de®ne accurately the capability and tolerance of
* Corresponding author. Tel.: +181-258-2839; fax: +181-2582700. E-mail address: [email protected] (J. Seghatchian).
new ®lters or ®ltration process in the routine production setting. It should also be practical and lend itself to standardisation and large scale screening to deal with the possibility of high frequency testing due to non-conformance subsequent to ongoing process validation and ®nal product quality monitoring. This national evaluation to assess the suitability of the IMAGN 2000, a Static Microvolume Fluorimetry, for leucocyte counting in red cell and platelet concentrates was carried out at three sites representing one site for each of the three zones. Comparative analyses were performed with the ¯ow cytometry procedures in the three dierent NBS laboratories in order to obtain evidencebased information into the problems and limitations of each analytical technology as well as the possible intersite variabilities. A limited number of samples were also analysed by microscopic technique using a large chamber (standard Nageotte) currently used as a ``Gold Standard''. However, it is fair to say that a manual method such as Nageotte, cannot be considered as a practical alternative due to a high intersite variability for counting the residual leucocytes in the leucodepleted products, the increased testing requirements and the limited availability of labour resources and technical skill.
0955-3886/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 5 - 3 8 8 6 ( 0 0 ) 0 0 0 1 8 - 7
78
J. Seghatchian et al. / Transfusion Science 22 (2000) 77±79
2. Material and methods
3.2. Platelet products
A total of 520 ®ltered red cell units locally produced were analysed in parallel by IMAGN 2000, ¯ow cytometry and at random by Nageotte chambers. The ¯ow cytometry procedures used by the three sites were not standardised against the same reference material although they were all based on the same principles of cell ®xation/permeabilisation followed by propodium iodine (PI) staining. The absolute counts were obtained by using reference ¯uorescent beads. In addition, a total of 353 platelet products prepared by locally existing leucodepletion procedures were also analysed in parallel by the three methods. All samples were tested within 36 h of donation by designated QM individuals, using their existing SOP. Various statistical analyses were performed by Biometric Imaging/Becton Dickinson on behalf of NBS as well as independently by each site.
The mean IMAGN values for the three sites (A, B and C, respectively) were 0.48, 2.75 and 2.44 WBC/lL; the mean ¯ow cytometry values were 1.33, 2.97 and 4.74 WBC/lL. Sites A and B cytometric values were 0:0=lL for 27% of red cells tested whereas none of the results in site C were reported as zero values. None of the 353 samples (all sites combined) gave a zero value by IMAGN. 28/353 units had 1.0 to 5:0 106 cells/unit as de®ned by the IMAGN method compared to 47/353 units analysed by ¯ow cytometry. Bland±Altman plots indicated a mean inter-method bias of +0:37 106 cells/unit for ¯ow cytometry results with signi®cant discrepancies outside the 95% limits of agreement being associated with higher estimates by ¯ow cytometry. At the higher decision point of 5:0 106 cells/units, twelve failures (out of 353 tested) were agreed by both ¯ow cytometric and IMAGN. There were additionally three interpretative discrepancies at the 5:0 106 level; only one was tested in parallel by Nageotte; this agreed with the IMAGN results. Bland±Altman plots of Nageotte and IMAGN data revealed no sign®cant inter-method bias. Nageotte and ¯ow cytometry data comparisons indicated that major outliers were due to signi®cant overestimates by ¯ow cytometry.
3. Results 3.1. Red cell products The mean IMAGN values for the three sites (A, B and C, respectively) were 1.26, 1.46 and 1.18 WBC/lL; the mean ¯ow cytometric values were 1.09, 1.48 and 2.72 WBC/lL. Sites A and B ¯ow cytometric values were 0.0/lL for about 30% of red cells tested whereas only 3% of results in site C were reported as zero values. IMAGN zero values were uniformly 3% for all three sites. 21/520 units had 1.0 to 5:0 106 cells/unit as de®ned by the IMAGN method compared to 74/520 units analysed by ¯ow cytometric. Bland±Altman plots indicated little inter-method bias between ¯ow cytometry and IMAGN results although only a small number of samples gave signi®cantly higher ¯ow cytometric values than IMAGN. The discrepancies between ¯ow cytometry and IMAGN were mainly restricted to the <2:0 106 level and were primarily associated with one site. At the higher decision point of 5:0 106 cells/unit, 1/520 units had >5:0 106 cells/unit by the IMAGN method and 2/520 units had >5:0 106 cells/unit by ¯ow cytometry.
3.3. Non-conformers In parallel some investigations were carried out on both red cell and platelet products showing recurrent failures and abnormal leucocyte distribution patterns as determined by ¯ow cytometry. While overall good agreement was observed between methods on platelet samples, discrepant results were identi®ed on some ®ltered red cell concentrates, supporting our earlier ®ndings on red cell leucodepleted product analysed locally [1]. The results also highlight the limitation of nonstandardised reagents in ¯ow methods with respect to the lack of some inter-site agreement and consistency. In this respect further work is needed to assess inter-changeability of data for all types of product.
J. Seghatchian et al. / Transfusion Science 22 (2000) 77±79
4. Conclusion This study represents the largest evaluation yet undertaken into the performance of IMAGN 2000. The results con®rm that there is a very good level of general agreement between ¯ow cytometry and IMAGN leucocyte counts on the same samples, but some samples show higher leucocyte counts by ¯ow cytometry as compared to both IMAGN and Nageotte. The results also highlight the limitation of nonstandardised reagents and gate setting, etc. in ¯ow cytometric methods which might contribute to lack in some intersite variability. In this respect additional work is in progress to establish a common protocol using a validated and standardised
79
reagent. Hopefully this would allow interchangeability of data for all types of products on all sites.
Acknowledgements We wish to thank Steve Scott for providing the statistical analysis for preparation of this manuscript.
Reference [1] Krailadsiri P, Seghatchian J. Evaluation of IMAGN 2000: a new system for absolute leucocyte count. Transfusion Science 1998;19:405±7.
www.elsevier.com/locate/transci
National evaluation of IMAGN 2000 for quality monitoring of leucodepleted red cell and platelet concentrates: comparison with ¯ow and Nageotte J. Seghatchian a,*, P. Krailadsiri a, B. Chandegra a, M. Beard a, N. Beckman b, L. Bissett b, B. Morris c, W. Booker c a
National Blood Transfusion Service, Pro®cency Testing Laboratory, London & South East, UK b National Blood Transfusion Service, Birmingham, UK c National Blood Transfusion Service, Sheeld, UK
1. Introduction The urgent need for the implementation of universal leucodepletion of blood components in UK has led to rapid national evaluation of various types of commercially available ®ltration technologies for eective removal of leucocyte in blood components. Prerequisite to achieve this objective is the establishment of a precise and standardised methodology, in various production sites, for sampling and counting the residual leucocytes. To be of relevance the measurement should be carried out on ``real life'' samples, without additional processing, to re¯ect the true nature of the product for clinical use. The method of choice for leucocyte counting must be highly sensitive to small changes in the ``decision making point'', i.e., WBC <1 106 and <5 106 /unit both to allow improvements in leuco®ltration technology, and to de®ne accurately the capability and tolerance of
* Corresponding author. Tel.: +181-258-2839; fax: +181-2582700. E-mail address: [email protected] (J. Seghatchian).
new ®lters or ®ltration process in the routine production setting. It should also be practical and lend itself to standardisation and large scale screening to deal with the possibility of high frequency testing due to non-conformance subsequent to ongoing process validation and ®nal product quality monitoring. This national evaluation to assess the suitability of the IMAGN 2000, a Static Microvolume Fluorimetry, for leucocyte counting in red cell and platelet concentrates was carried out at three sites representing one site for each of the three zones. Comparative analyses were performed with the ¯ow cytometry procedures in the three dierent NBS laboratories in order to obtain evidencebased information into the problems and limitations of each analytical technology as well as the possible intersite variabilities. A limited number of samples were also analysed by microscopic technique using a large chamber (standard Nageotte) currently used as a ``Gold Standard''. However, it is fair to say that a manual method such as Nageotte, cannot be considered as a practical alternative due to a high intersite variability for counting the residual leucocytes in the leucodepleted products, the increased testing requirements and the limited availability of labour resources and technical skill.
0955-3886/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 5 - 3 8 8 6 ( 0 0 ) 0 0 0 1 8 - 7
78
J. Seghatchian et al. / Transfusion Science 22 (2000) 77±79
2. Material and methods
3.2. Platelet products
A total of 520 ®ltered red cell units locally produced were analysed in parallel by IMAGN 2000, ¯ow cytometry and at random by Nageotte chambers. The ¯ow cytometry procedures used by the three sites were not standardised against the same reference material although they were all based on the same principles of cell ®xation/permeabilisation followed by propodium iodine (PI) staining. The absolute counts were obtained by using reference ¯uorescent beads. In addition, a total of 353 platelet products prepared by locally existing leucodepletion procedures were also analysed in parallel by the three methods. All samples were tested within 36 h of donation by designated QM individuals, using their existing SOP. Various statistical analyses were performed by Biometric Imaging/Becton Dickinson on behalf of NBS as well as independently by each site.
The mean IMAGN values for the three sites (A, B and C, respectively) were 0.48, 2.75 and 2.44 WBC/lL; the mean ¯ow cytometry values were 1.33, 2.97 and 4.74 WBC/lL. Sites A and B cytometric values were 0:0=lL for 27% of red cells tested whereas none of the results in site C were reported as zero values. None of the 353 samples (all sites combined) gave a zero value by IMAGN. 28/353 units had 1.0 to 5:0 106 cells/unit as de®ned by the IMAGN method compared to 47/353 units analysed by ¯ow cytometry. Bland±Altman plots indicated a mean inter-method bias of +0:37 106 cells/unit for ¯ow cytometry results with signi®cant discrepancies outside the 95% limits of agreement being associated with higher estimates by ¯ow cytometry. At the higher decision point of 5:0 106 cells/units, twelve failures (out of 353 tested) were agreed by both ¯ow cytometric and IMAGN. There were additionally three interpretative discrepancies at the 5:0 106 level; only one was tested in parallel by Nageotte; this agreed with the IMAGN results. Bland±Altman plots of Nageotte and IMAGN data revealed no sign®cant inter-method bias. Nageotte and ¯ow cytometry data comparisons indicated that major outliers were due to signi®cant overestimates by ¯ow cytometry.
3. Results 3.1. Red cell products The mean IMAGN values for the three sites (A, B and C, respectively) were 1.26, 1.46 and 1.18 WBC/lL; the mean ¯ow cytometric values were 1.09, 1.48 and 2.72 WBC/lL. Sites A and B ¯ow cytometric values were 0.0/lL for about 30% of red cells tested whereas only 3% of results in site C were reported as zero values. IMAGN zero values were uniformly 3% for all three sites. 21/520 units had 1.0 to 5:0 106 cells/unit as de®ned by the IMAGN method compared to 74/520 units analysed by ¯ow cytometric. Bland±Altman plots indicated little inter-method bias between ¯ow cytometry and IMAGN results although only a small number of samples gave signi®cantly higher ¯ow cytometric values than IMAGN. The discrepancies between ¯ow cytometry and IMAGN were mainly restricted to the <2:0 106 level and were primarily associated with one site. At the higher decision point of 5:0 106 cells/unit, 1/520 units had >5:0 106 cells/unit by the IMAGN method and 2/520 units had >5:0 106 cells/unit by ¯ow cytometry.
3.3. Non-conformers In parallel some investigations were carried out on both red cell and platelet products showing recurrent failures and abnormal leucocyte distribution patterns as determined by ¯ow cytometry. While overall good agreement was observed between methods on platelet samples, discrepant results were identi®ed on some ®ltered red cell concentrates, supporting our earlier ®ndings on red cell leucodepleted product analysed locally [1]. The results also highlight the limitation of nonstandardised reagents in ¯ow methods with respect to the lack of some inter-site agreement and consistency. In this respect further work is needed to assess inter-changeability of data for all types of product.
J. Seghatchian et al. / Transfusion Science 22 (2000) 77±79
4. Conclusion This study represents the largest evaluation yet undertaken into the performance of IMAGN 2000. The results con®rm that there is a very good level of general agreement between ¯ow cytometry and IMAGN leucocyte counts on the same samples, but some samples show higher leucocyte counts by ¯ow cytometry as compared to both IMAGN and Nageotte. The results also highlight the limitation of nonstandardised reagents and gate setting, etc. in ¯ow cytometric methods which might contribute to lack in some intersite variability. In this respect additional work is in progress to establish a common protocol using a validated and standardised
79
reagent. Hopefully this would allow interchangeability of data for all types of products on all sites.
Acknowledgements We wish to thank Steve Scott for providing the statistical analysis for preparation of this manuscript.
Reference [1] Krailadsiri P, Seghatchian J. Evaluation of IMAGN 2000: a new system for absolute leucocyte count. Transfusion Science 1998;19:405±7.