First invented by Cesar Milstein and George Kohler, the monoclonal antibody technology initially resulted in antisheep red blood cells immunoglobulins more reliable than the variable polyclonal mixtures produced by rabbits. This typically “home brewed” or laboratory developed test (LDT) reagent was rapidly followed by the worldwide production of an immense array of commercially produced, far superior preparations with exquisite specificity. Monoclonal antibodies were developed into a myriad of diagnostic reagents and, more recently, as powerful therapeutic tools.
The novelty of the concept and its perfect adaptability to laboratory testing has been at the root of the development of clinical flow cytometric immunophenotyping. Because of the versatility of these monoclonal antibody tools, flow cytometry has evolved as a refined technique which, properly used, can be applied to many types of explorations. The latter range from the qualitative identification of simple or complex immunophenotypes of cell subsets to the accurate detection and counting of minute numbers of rare events.
Flow cytometry is now well-mastered and applied daily in hematology and immunology laboratories. However, it is far from a fully mature technology and keeps evolving. For instance, the number of parameters liable to be assessed concomitantly keeps growing, thanks to ongoing progress in analysis software. A tremendous number of publications have thus appeared since the early 1980s with the emergence of this new field, and the number is still growing as new applications are continuously described. As a consequence, cell-based fluorescence assays are typically LDTs and thus require standard operating procedures with an adapted level of validation.
LDT validation, as understood by most accreditation agencies, usually requires the demonstration of such parameters as sensitivity, specificity, reproducibility, linearity, trueness, and stability, to indicate only a few. However, flow cytometry, at variance to other laboratory tests, has two intrinsic properties: (i) by definition, it examines thousands of events and parameters in each single test and (ii) it addresses a variety of clinical conditions which themselves are quite pleomorphic and potentially patient-specific. The paradigms used for other laboratory tests, therefore, cannot be straightforwardly transposed to the validation of flow cytometry LDTs. Yet, a number of safeguards must and can be satisfied in order to provide clinicians, and more importantly patients, with reliable and useful data.
Here, as explained in the Editorial Introduction to this Special Issue of Clinical Cytometry Part B, a group of experts has strived to produce what they hope (and the reviewers believed) will be a useful reference document for flow cytometry laboratories. It deals with all aspects of flow cytometric LDT development and masters multiple applications for this highly versatile and accurate methodology.
Marie-Christine Béné, PharmSciD, PhD
Immunology LaboratoryCHU de Nancy & Lorraine Université
Vandoeuvre les Nancy, France
My co-editor has sketched the history of monoclonal antibodies and their application to clinical flow cytometry and the goals of this special section. The history of how this Clinical Cytometry Special Section of Practice Guidelines for Validation in Clinical Cytometry came to be is well outlined in its Editorial Introduction. It is followed by four sections: (i) Pre-analytical Considerations, (ii) Analytical Issues with eight figures, (iii) Post-Analytic Considerations, and (iv) Assay Performance Criteria. It is important to note that this effort led by Bruce H. Davis, David Barnett, Brent L. Wood, and Teri Oldaker involves both the International Council for Standardization of Haematology (ICSH) and the International Clinical Cytometry Society (ICCS). Some of this material was introduced with initial discussions at a recent Center for Devices and Radiological Health (CDRH) FDA workshop on Clinical Flow Cytometry in Hematologic Malignancies (February 2013). An archival webcast, agenda with linked slides, and transcript are available at this link: http://www.fda.gov/MedicalDevices/NewsEvents/WorkshopsConferences/ucm334772.htm#webcast.
Reviewers were selected and asked to review the editorial and the four articles all as one document. This approach entailed a larger workload for each reviewer and the Special Editors and Editor-in-Chief are tremendously grateful for all of their efforts. It is noted that these guidelines are being offered as a consensus of two professional organizations.
What can be said about the review itself? All reviewers recognized the tremendous amount of consensual work done and the need for such a document, yet they all favored some degree of revision. Potential conflicts of interest were dealt with immediately and are listed in the document. One reviewer stressed the need to differentiate between “validation and practice guidelines,” and in fact, the final document may be more representative of current practice than a document for validation. This can sometimes be seen as the difference between an evidence-based position versus personal experience. It was pointed out that the role of morphology applies almost uniquely to leukemias and lymphoma and not to lymphocyte subset analysis, MRD detection or CD34 counting. It was suggested that the authors separate laboratory accreditation from validation and from practice guidelines, and it was noted that most pathologists are not trained to analyze list-mode data.
There was discussion about the number of events needed to define a cluster- 100 cells versus 20–30. This of course included the definition of the denominator to be used and what numbers are to be reported. There was a recommendation for a literature review on MRD in hematologic malignancies. In fact, the Center for Drug Evaluation and Research at FDA has sponsored and conducted three workshops on minimal residual disease (MRD) as a surrogate endpoint in acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL) and acute myelogenous leukemia (AML). The need for software review was suggested. And, Brent Wood led a major CDRH-FDA workshop session on this topic.
Two extra articles have been included in this Special Issue. The first is by de Carvalho Bittencourt et al. and is entitled “Value Of HIV Patients With Regular Follow-Up As In-House Internal Controls Of Flow Cytometry Measurement Of Lymphocyte Subsets.” This article generated a “Controversies in Clinical Cytometry.” There are two accompanying letters- one “supporting” and the other “disagreeing” with this article and the readership is left to decide its applicability. The second article by Davis et al. is entitled “Determination of Optimal Replicate Number for Validation of Imprecision using Fluorescence Cell Based Assays: Proposed Practical Approach.” The review of this article also resulted in controversy concerning the development of a novel, data-derived variance factor to determine the precision of an assay. Again, it will be left to the reader to decide the validity of this approach, both in terms of further critical commentary on this article and its eventual replication in another laboratory.
Gerald E Marti, MD, PhD, Medical Officer
Heme/Path Branch, Flow Cytometry andImmunology Branch
Division of Immunology and Hematology Devices
Office of In Vitro Diagnostic and Radiologic DeviceEvaluation and Safety (OIR)
Center for Devices and Radiologic Health (CDRH)
Food and Drug Administration
PS. Both guest editors would like to thank Frederic Preffer, PhD, Editor-in-Chief and Doris A. Regal, Executive Secretary at Cytometry Part B, Clinical Cytometry, for their assistance in this endeavor. Besides the usual difficulties of dealing with guest editors, the offices of the Journal had recently moved from Chicago to Boston while we were preparing this Special Issue causing additional problems primarily for Ms. Regal, which she handled superbly! Thank you both.