Cytometry Part B (Clinical Cytometry) 82B:9–17 (2012)
Original Article
ISHAGE Protocol: Are We Doing It Correctly? Alison Whitby,1 Liam Whitby,1 Matthew Fletcher,1 John T. Reilly,1 D. Robert Sutherland,2 Michael Keeney,3 and David Barnett1* 1
UK NEQAS for Leucocyte Immunophenotyping, Department of Haematology, Royal Hallamshire Hospital, Sheffield S10 2QN 2 Department of Laboratory Hematology, University Health Network, Toronto, Canada 3 Hematology/Flow Cytometry, London Health Sciences Centre, London, Canada
Background: Flow cytometric CD341 stem cell enumeration is routinely performed to optimize timing of peripheral blood stem cell collections and assess engraftment capability of the apheresis product. While a number of different flow methodologies have been described, the highly standardized ISHAGE protocol is currently the most widely employed, with 204/255 (81%) international participants in the UK NEQAS CD341 stem cell enumeration program indicating their use of this method. Recently, two laboratories were identified as persistent poor performers, a fact attributed to incorrect ISHAGE protocol usage/ setup. This prompted UK NEQAS to question whether other laboratories were making similar errors and, if so, how this might affect individual EQA performance. Methods and Results: In send out 0801, where two stabilized samples were issued, the EQA center surveyed 255 participants with flow analysis data and subsequent results collected. One hundred and ninety-six laboratories returned results with 103 returning dot plots. Eighty-three out of one hundred and three stated that they used the ISHAGE protocol gating strategy but 43% (36/83) were incorrectly setup. Analysis of the data showed those incorrectly using single platform ISHAGE gating strategy were twice as likely to fail an EQA exercise compared to those using the protocol correctly. This failure rate increased two fold when incorrect ISHAGE protocol was used in a dual platform setting. Conclusion: This study suggests a widespread fundamental lack of understanding of the ISHAGE protocol and the need to deploy it correctly, potentially having significant clinical implications and highlights the need to monitor participants rigorously in their deployment of the ISHAGE protocol. It is hoped that once these findings have been disseminated, performance can be improved. VC 2011 International Clinical Cytometry Society
Key terms: CD34; stem cells; ISHAGE; quality control; quality assessment; EQA
How to cite this article: Whitby A, Whitby L, Fletcher M, Reilly JT, Sutherland DR, Keeney M, Barnett D. ISHAGE protocol: Are we doing it correctly? Cytometry Part B 2012; 82B: 9–17.
Over the last 15 years or so, cytokine-mobilized peripheral blood stem cells (PBSC) have largely replaced bone marrow as a source of hematopoietic stem cells (HSCs) in the majority of autologous and (in an increasing proportion) allogeneic PBSC transplants (1,2). The HSCs in marrow and peripheral blood, which are responsible for multilineage engraftment in the transplant setting express the cell surface marker CD34 (3,4). Flow cytometric enumeration of CD34þ cells provides a rapid means of measuring this clinically useful surrogate marker of graft adequacy in all sources of HSCs and most transplant centers now determine graft adequacy based on the number of CD34þ cells/Kg of patient body
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weight. In addition to determining yield, the number of CD34þ cells mobilized to the peripheral blood is also a predictor of the success of apheresis (5) and can be used to monitor, ‘‘on-line,’’ the yield of CD34þ cells (6). *Correspondence to: Dr. David Barnett, Deputy Director and Consultant Clinical Scientist, UK NEQAS for Leucocyte Immunophenotyping, 4th Floor, Pegasus House, 463a Glossop Road, Sheffield S10 2QD, England. E-mail:
[email protected] Received 18 March 2011; Revision 6 July 2011; Accepted 8 July 2011 Published online 19 July 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/cyto.b.20612
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Table 1 Gating Strategies Used by all Participants in the UK NEQAS for Leucocyte Immunophenotyping CD34þ Stem Cell Enumeration program, Compared to Those Used by the 103 Participants That Returned Dot Plots for the Study Gating strategy ISHAGE STEMKIT PROCOUNT MILAN BENDER
Study participants (%)
All particpants (%)
83 4 5 5 1
81 8 4 6 1
Although simple flow methods for counting CD34þ cells date back to the late 1980’s (7–13), a standardized multi-parameter protocol that was based on the structural characteristics of the CD34 molecule and the epitopes detected by various CD34 monoclonal antibodies did not emerge until 1994 (14). The latter used the maximum information available of four parameters; forward and side light scatter and the intensity of CD34 and CD45 staining and subsequently formed the basis of a Clinical Guideline for CD34þ cell quantitation in peripheral blood (PB) and PBSC for the International Society for Hematotherapy and Graft Engineering (ISHAGE) (15). Subsequent developments included the incorporation of a viability dye 7-Amino Actinomycin D (7-AAD) and fluorescent counting beads, in a Single Platform (SP) variant (16). The addition of counting beads eliminates the potential introduction of errors in calculating the absolute CD34þ cell count inherent in two platform (cytometer plus hematology analyzer) methodologies (16–18). The essential components and technical details of the ‘‘SP ISHAGE with viability Protocol’’ are embodied in several National (19,20), Regional (21), and International Guidelines (22–24). The single platform methodology has been validated on a wide range of instruments employing a variety of different types of fluorescent counting beads (25). The use of SP flow cytometric technology has improved standardization globally of this procedure (18). Indeed, using the SP ISHAGE approach, CVs of
