Guidelines on the use of multicolour flow cytometry in the diagnosis of haematological neoplasms

Please refer to Appendix I for classification of evidence levels. Standard operating procedures for all areas of the laboratory require validation. These and all controlled documents may be audited; in particular: The guideline group comprised representatives of UK-based clinical flow cytometry (FC) experts and clinical representatives. The writing group produced the draft guidelines, which were subsequently revised by consensus by members of the Haemato-oncology Task Force of the British Committee for Standards in Haematology (BCSH). The guidelines were then reviewed by a sounding board of approximately 50 UK Haematologists and FC experts, the BCSH and the British Society for Haematology (BSH) Committee, and comments incorporated where appropriate. Significant technical advances have taken place in the field of flow cytometry (FC) since 2002, when the BSCH last published FC guidelines. Cytometers with three or more lasers are now common in routine clinical practice and the number of commercially available fluorochromes has increased rapidly. This enables the simultaneous analysis of eight or more antigens, so-called multicolour (or polychromatic) flow cytometry (MFC). In addition, modern cytometers analyse larger numbers of cells in a shorter time, and new software is changing the way data is or can be analysed. This new level of complexity demands specialist knowledge and technical expertise if it is to yield accurate and reproducible results (Sewell & Smith, 2011). Application of MFC affects nearly all areas of clinical FC practice, including instrument optimization, quality control (QC), panel design, sample preparation, analysis and interpretation, reporting, and training. The aim of this document is to provide healthcare professionals with clear guidance on the use of MFC for leukaemia and lymphoma immunophenotyping of peripheral blood (PB), bone marrow (BM), body fluids and tissue specimens. In Vitro Diagnostic Medical Device (IVD) European Compliant (CE) instruments come fully specified, with lasers and filters designed for optimal analysis of the manufacturer's own and most common fluorochromes. If these cytometers are used in other conformations, for example with different filters, they lose their CE-IVD compliance. Other cytometers allow a choice of lasers and filters. For example, a 532 nm laser may be selected over a 488 nm laser, as the former results in less autofluorescence (Perfetto & Roederer, 2007). It is strongly recommended that lasers with known good stability and suitable for use with available reagents be chosen. It is imperative to ensure that the filters used are suitable for the detection of the reagents used. Several web resources are available that show the laser output together with the excitation and emission spectra of commercially available fluorochromes (Appendix II). Use of non-CE-IVD equipment will not prevent laboratory accreditation provided that the relevant validations are carried out together with the use of internal controls, and that the laboratory participates in suitable external quality assurance schemes. The performance and operation of an instrument cannot be predicted from the laser and optics specifications alone; therefore, a trial period is strongly recommended before a commitment to buy an instrument. Availability of product specialist support and engineering support are also important factors to consider. A system optimization set-up that determines the optimal photomultiplier tube (PMT) voltages is vital to achieve best instrument performance and is required at system set-up, after installation of new lasers or laser fibres, and after significant realignment of lasers or installation of new optical filters (Perfetto This can be if and the use of software is recommended the system optimization, and for new fluorochromes or it is strongly recommended that be to allow This can be by software that it may be to for example, for for or as a of This can be by the or the for the cells of over a of different voltages & voltages not on the or group antigens, as on cells or on These may require if the are to or fluorochromes. For the most of and of as as of the for for is and the aim be to not or this the can in of and in on the so-called the of all is not known to the to voltages Application & on cells it is recommended that is on It is recommended that be as these allow all to be on the software have a or have other or It is to be with the data is and that the may be on the used It is recommended that for and be to achieve a clear and of the of (or of on the be In clinical the of may in the of to use a for software has to and a good analysis and of be or and are recommended for analysis of cells and of analysis of in may as of and cytometers allow the to the at which a is more the where a the This so-called may be as of the set-up or on (Perfetto If be to the of the (Perfetto It is recommended that a be used for all If for this is not to is a control system to that the and filters are the to be for if the laboratory software for of voltages and other is for instrument It a system which laser and the for instruments that use this This is if are available that a set-up & all instruments have software that laser and the manufacturer's is to laser as an or control that allow data to be a and and by a product specialist are by it not be to on a It may be for performance of cytometers of the and to achieve optimal instrument The instrument be and not be by or This that and are and voltages are to an if a Standard are that with of in all used. The voltages can then be on a that the to the The use of software to this is strongly for It is to the on a of cytometers that the on a sample are writing group This is to as and may be for trial or and that use the results from the cytometers are on including the number and different of cytometers In is by and to all It is to use a common and to that all cytometers their not be the optimal for in the group cytometers with different lasers and or filters are cells be used to the the are are with and can subsequently be used for the be cytometers of and have from the all from sample to also be is on the of the it is the or of that best the is to on the of cells in addition, and autofluorescence have also to be of 2011). practice is to use or for the and the of the the the and laser therefore, the more the the other the be to the from an is it is strongly recommended that all data are in a for and to be that can be It is that is the of results in are good web resources that the of and to (Appendix II). for eight or more can be the of and is MFC software for of and will It is strongly recommended that these are used. is strongly recommended when the be If is the factors can be if the In addition, immunophenotyping of leukaemia and lymphoma that may have different of and may not be fully for all a is an example of a so-called This by the and the data to be and the used to a to that be for all the in the sample if not cytometers of eight or more use and can be or after data in clinical flow a number of multicolour is the and known as or FC guidance or that and are used & & Roederer, 2007). This is for clinical FC to and and are used. For example, may be used for all and it be that not all of a are the and in addition, the different may also It is recommended that all be for to which require a These of the and be document The group of fluorochromes that require are the fluorochromes. These different and The and of in and with fluorochromes & & 2007). may be fluorochromes produced by different that of a & Roederer, This group has also different for fluorochromes from different In have significant in the of and for these a of and from the and to the & In addition, that use in a of may it to for new A to this may be the for new is to that of the If the is is not this demands instrument performance and on knowledge and of the can before different If instrument stability is not the relevant validations for are by in have not it is recommended that the is when new of be to the in are and and good on the other may in their of and therefore, they may are and of the and used is are at where cells may results with to and cells have different in laser to most therefore, cells be used with to fluorochromes & 2011). of which to be for in different of to or cells and cells may the best are cells to be used when for or a a multicolour tube set-up may use a of cells and the control for the as the control & Roederer, 2007). For for a multicolour tube that and use cells and The cells require cells as and the require or as The aim of routine is to in to an accurate and The of to and fluorochromes to with will on of and a panel has it be by the in this Several guidelines for the of & 2011). In addition, the of of and important together with their and other clinical and Appendix that for and The when to in MFC The be different for a are commercially will be known and published and most are if a in a results may when it is used as of a of be on known to for clinical analysis of as as on their in the of the multicolour that they are used. be selected on the lasers and filters to be for example, and be to use in the Several show spectra for most commercially available to be of these are in Appendix The of fluorochromes for the different has example, & Roederer, 2007). a fluorochromes are used for detection of and for and the of also be the they are which affects the of the For example, a as to will the If the used on is by the if it is more may be It can be to a will a for is an It is to have to an optimal and to ensure that all as with to of In a of be and may have to be used in a less of an example where an important is used on a and to may analysis for the less In a to the may be on In this the cells and used on In a a may have a in the this the cells were This tube be by the of and by or on a and in a that is not by This has in & Roederer, 2007). The be The by and the tube with all of the tube to be then a of of the at a Appendix of and when to use a has selected and the in different of to be This is of for data that can and is used. if software is it is strongly recommended that all and have good knowledge of for the used and as as and be that internal and control as as The as by are important as they provide vital for from and an of to the This knowledge to different sample as on the In addition, that to new fluorochromes for to new to cells at at and it is strongly recommended that a of from is For example, when leukaemia from may be used as be in the at from is important for as is to & & for cytometry it is important to be of the and use to all available of all is strongly recommended 2007). The of reagents in a laboratory can be a of In addition, of reagents from the and to will their or are are use or that from are used. may be or of that is to be as a a validation. may also be reagents that are not suitable for in a for the are to in their and may at & The of this writing group is that eight or more to including has in of A of for the preparation, and document control required for is clinical and or blood be with the immunophenotyping sample to allow for optimal of in with flow analysis is if a panel is used and the clinical are The of fully is in for and guidance on 2011). routine may be required to This is by by an or from with increased of system be for FC of as this is to be more and quality be to for example, by of a from the and cells may analysis to of and group The of in clinical are or other of are not to A is tissue are for or cells may the of cells & are for of and may the analysis is by or for tissue where is a be used in MFC as be to or from relevant as they are from in the of may be from a of cells in as sample not be as The use of may be for analysis where more of cells is as and for the of Several reagents are of on their in the of and and and are suitable for MFC analysis of and 2011). are and with all these published guidelines for clinical FC in For analysis the of and in for or in for is recommended For is For or is A of this can be in Appendix In with BCSH guidelines this as it enables The use of FC has and since the revised BCSH immunophenotyping guidelines are more and from in tissue group It is strongly recommended to the use of in as this may autofluorescence are also available and are for as these analysis are not suitable for is also more in the of are known to be by and & & and and at and to they are if at to group in the of control and the when and it is to show that are not by that be to analyse and sample is at it may be to all for at is strongly recommended for For of or have not in of of the most common or for to group the for and is for a of and of at for to are recommended may be selected for this be by Appendix and of for example, a from a that is for It is an important that are not by the of the optimization, and and the different fluorochromes other also determines the The of the MFC the to from is to all of these not as to these will and it is recommended that laboratory in sample be carried out and be for tissue to may the of and may also fluorochromes If is it is strongly recommended that be to their use in The be at that are before to or is recommended over the of for where or and is required to & European it is to cells after so-called This group and be to A of be used as a or may in after is recommended to to this where is used. For example, or and blood all a increased number of the of cells as cells after is For after is recommended is a of cells and of to is group It be that by a of as as a of and group This may the for FC where are at to can be by of all a as the and be the aim The of cells tube on the more cells will be for or In and may group A at in this writing The the the more will to the best of is published evidence to show that on at or for a with or is an may be at a to and be taken to will be where are This results or internal controls, if these have a with autofluorescence as and may be used. are not recommended for routine analysis and not be used to cells If used for as they the all the in the tube be A of for to control for published The in that are by this writing and are incorporated in the and this on and their can be in Appendix and of is for all data this has to be in a that for data of sample flow software will allow for data to be and different and or This may be analysis in to it be a of The number of to on sample and and It be that an number of are for the cells and internal For example, of may at 50 of all The number of required to a and of For or for may be the and use of to cells have be taken to of the of The of FC data for at of the of on the number of 2007). For or are used a the of analysis that leukaemia and not If are carried it is that MFC will to be on the A or is recommended as This stability and enables on data if a has be taken to may in increased of and which results in and In addition, increased at has in with for of of or all or recommended for cells and has in the of clinical published data is available on the of on clinical MFC data for different cytometers It is recommended that laboratory the of flow on their be and as FC with These are by the if not all modern FC analysis software & The data of the set-up including and in to a of and may not the used for analysis and reporting, as it is to this at will over time, for example, with of and are not on the cytometers can and it is recommended that used are document The be of the data stability of and of internal If all these are the data can be for the of if and including or to the are used to in on of The choice of on and software used and with the number of used. It is recommended that a be used that as as relevant or so-called Appendix for MFC in can have of and The of these can be as in a (or or in a & For lymphoma where the have of it is recommended that the of or all the The of the cells can be from a The analysis is also the in for For all and for or analysis in it is important to that an or are not by A and of of the cells of in all and is It is and important to show that that are for are for at other that not show a in or in be have published UK guidelines or on to FC immunophenotyping In addition, the of and new software results in more data and more can be more and more The factors for this be by that show of MFC For where a may or for of in of with The of MFC data and analysis is to and this may are software have at that a for of These may be which may not be as the flow of an & the of The are at a of MFC can be in Appendix MFC is and technical expertise and members have not used MFC will MFC if they have FC and In the areas of and data analysis and data require training. and of for all the clinical FC laboratory are This will in the level and of required for of and that and reproducible results are be and the It is recommended that these are reviewed on an and web resources may be for members are recommended to in MFC and to external FC The for good MFC knowledge in to accurate results not be and that on MFC are suitable for In addition, that on clinical with an on analysis and interpretation, also be with and have a to new clinical a of with a of is for flow laboratory and a of resources can be in Appendix The of MFC will to new areas that require and (Perfetto & 2011). These have this for of a of the areas recommended for is Standard operating procedures for all areas of the laboratory require validation. These and all controlled documents may be audited; in particular: the and in these guidelines is to be and accurate at the of to the the British Society for Haematology the for the of these guidelines. guidelines for the or of multicolour flow cytometry have guidelines for immunophenotyping of leukaemia Haematology Task Force of the and Haematology Task Force of the were revised in These new guidelines are designed to these and to provide on technical and training. of the have a of and to this by and and The to and for technical and and and for of and are when is that the or not and can be to most as the of or not is less a is require to as The quality of evidence is as or this in it is to the of knowledge and or is to in the of evidence from clinical important may have an important on in the of and may the evidence from clinical with important or of to to to to or evidence from or or and of the of a or of a is to have an important on in the of and is to the evidence from or It is a that all MFC have and to their level of achieve a clear of and their is The are on writing and the consensus for clinical FC and 2007). In addition, it is recommended that be blood This and sample and In addition, & including and sample be of A preparation, and sample knowledge of and sample quality or with instrument is of relevant for all and and is also and sample data analysis can be results be by a of in a that if the may to of and A in a that in MFC and external in these areas is and This group will be for instrument optimization, and data knowledge of sample and is Diagnostic will be in for leukaemia and be to and from of This expertise is by on the including data analysis of known and be to leukaemia results to It is best practice for to have of new For more analysis and it is that the is reviewed with to the as analysis and reporting, a of in to they also be with analysis software will provide and for healthcare after a of clinical MFC and This will have for the FC The demands knowledge of MFC and in to the technical and knowledge of all of the and MFC will be in with and other laboratory and is This is by that on and with This group be for the flow with a of the will then be of the at the that is and will have in a including external and is The of and will on available and is to the most important are that instrument optimization, and not be by to and with require in MFC data and MFC and instrument is provided by this is not for that have a in a FC of the of the in of be that to of are in 2011). A to is with the aim of of & 2011). The of an is to have to the and the for in for this to will to be and to and these as as a with clinical A with guidelines for be that provide MFC that clinical resources for from new MFC tube be they are as published are The analysis of the data is it may a to be all other the are then for as or in different or in are also for may require use of the or a new be If a has an for a a be an tube where the for the is It is to on sample to for A is to the of the and of For most a good quality a of and and cells to have or have different with their It may be to have for at For example, that have larger or with be selected for In leukaemia has and it is to have an for the an may be and a or be to It may then be a and a tube with the The analysis of for the of is fluorochromes be by all In the analysis as it is to be used in the laboratory be and by all with analysis and are of the and as the that are an that is to be on or in the cells to be analysed. be to the as as number of and have the as the This is not from the group have different procedures the control and be from the to ensure that and are the The use of a of that in the used to the with the this is not where the use of may be If the and all the in the tube be In a of cells will be cells that and not a of that in an are to as internal The internal control has to to the of including to the to of are to The control be selected on and to the For example, a sample have as internal control for common in leukaemia cells may have different from and not sample a as as a of the a is not or may be control is a of and an of the It is used to the of In and of the will be by the of If the control that in the sample of an be blood may be from a and are as they are to the available are also These have the of increased and most of as or or are for of and for as and of new of is the recommended for routine blood and more after or & is used as an for use has also recommended for flow of and together with as to for with for to of the of may for to in Several have that best for and & & is not suitable for and level of and increased on & published guidelines for clinical FC in For analysis the of and in for or in for is recommended For is For or is This for that the group has The will be as eight have published to This group cannot that of these published has as in these the used have by In addition, a of the published on for and are It is the of this document to the of The published for all in the of which were published The for for in 2007). for published by A of used by for can be in and A panel used in a more can be in for in published by for published by The use of by of for analysis can be in The use of and for of by published by were subsequently published by The European group for the use of FC in published panel and in and can be in The European published consensus for and for and on these can be for example, and for can be in These and for and for were published in the guidelines The is not the of in are published panel to the best of A for the use of for cells in and marrow can be in