Summary of validation considerations with real-life examples using both qualitative and semiquantitative flow cytometry assays.

In the clinical laboratory, flow cytometry assays are critical to providing diagnostic and prognostic information to the treating clinicians. A validation or verification provides confidence that the assay will yield reliable results that can be trusted to make critical medical decisions. The following performance specifications should be included in a validation for laboratory developed tests as needed: accuracy (or trueness), precision (reproducibility and repeatability), detection capability, selectivity, reference range, and sample and reagent stability. We define these terms and present our approach to validation of several common flow cytometry assays, including examples of a leukemia/lymphoma assay and a paroxysmal nocturnal hemoglobinuria (PNH) assay.

Flow Cytometry Method Validation Protocols.

Analytical method validation provides a means to ensure that data are credible and reproducible. This article will provide a brief introduction to analytical method validation as applied to cellular analysis by flow cytometry, along with practical procedures for four different types of validation. The first, Basic Protocol 1 (the limited validation protocol), is recommended for research and non-regulated laboratories. Next, Basic Protocol 2) presents a reasonable, fit-for-purpose validation approach appropriate for biopharma and research settings. Basic Protocol 3 addresses the type of validation performed in clinical laboratories for moderate-risk tests developed in house. Finally, Basic Protocol 4 describes the process that should be applied whenever a method is being transferred from one facility to another. All four validation plans follow the fit-for-purpose validation approach, in which the validation parameters are selected based on the intended use of the assay. These validation protocols represent the minimal requirement and may not be applicable for every intended use such as high-risk clinical assays or data to be used as a primary endpoint in a clinical trial. The recommendations presented here are consistent with the white papers published by the American Association of Pharmaceutical Scientists and the International Clinical Cytometry Society, as well as with Clinical Laboratory Standards Institute Guideline H62: Validation of Assays Performed by Flow Cytometry (CLSI, 2021). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Limited validation Basic Protocol 2: Fit-for-purpose validation for biopharma and research settings Basic Protocol 3: Validation for moderate clinical risk laboratory developed tests Basic Protocol 4: Transfer validation.

High-sensitivity flow cytometric assays: Considerations for design control and analytical validation for identification of Rare events.

The current consensus recommendation papers dealing with the unique requirements for the analytical validation of assays performed by flow cytometry address the validation of sensitivity (both analytical and functional) only in general terms. In this paper, a detailed approach for designing and validating the sensitivity of rare event methods is described. The impact of panel design and optimization on the lower limit of quantification (LLOQ) and suggestions for reporting data near, or below, the LLOQ are addressed. This paper serves to provide best practices for the development, optimization, and analytical validation of flow cytometric assays designed to assess rare events. Note that this paper does not discuss clinical sensitivity validation, which addresses the positive and negative predictive value of the test result.

Flow cytometric method transfer: Recommendations for best practice.

As with many aspects of the validation and monitoring of flow cytometric methods, the method transfer processes and acceptance criteria described for other technologies are not fully applicable. This is due to the complexity of the highly configurable instrumentation, the complexity of cellular measurands, the lack of qualified reference materials for most assays, and limited specimen stability. There are multiple reasons for initiating a method transfer, multiple regulatory settings, and multiple context of use. All of these factors influence the specific requirements for the method transfer. This recommendation paper describes the considerations and best practices for the transfer of flow cytometric methods and provides individual case studies as examples. In addition, the manuscript emphasizes the importance of appropriately conducting a method transfer on data reliability.

Flow Cytometry Method Validation Protocols.

Analytical method validation provides a means to ensure that data are credible and reproducible. This unit will provide a brief introduction to analytical method validation as applied to cellular analysis by flow cytometry. In addition, the unit will provide practical procedures for three different types of validation. The first is a limited validation protocol that is applicable for research settings and non-regulated laboratories. The second is validation protocol that presents the minimum validation requirements in regulated laboratories. The third is a transfer validation protocol to be used when methods are transferred between laboratories. The recommendations presented in this unit are consistent with the white papers published by the American Association of Pharmaceutical Scientists and the International Clinical Cytometry Society, as well as with Clinical Laboratory Standards Institute Guideline H62: Validation of Assays Performed by Flow Cytometry (currently in preparation). © 2018 by John Wiley & Sons, Inc.

ICCS/ESCCA consensus guidelines to detect GPI-deficient cells in paroxysmal nocturnal hemoglobinuria (PNH) and related disorders part 4 - assay validation and quality assurance.

Over the past six years, a diverse group of stakeholders have put forth recommendations regarding the analytical validation of flow cytometric methods and described in detail the differences between cell-based and traditional soluble analyte assay validations. This manuscript is based on these general recommendations as well as the published experience of experts in the area of PNH testing. The goal is to provide practical assay-specific guidelines for the validation of high-sensitivity flow cytometric PNH assays. Examples of the reports and validation data described herein are provided in Supporting Information. © 2017 International Clinical Cytometry Society.

2006 Bethesda International Consensus recommendations on the immunophenotypic analysis of hematolymphoid neoplasia by flow cytometry: recommendations for training and education to perform clinical flow cytometry.

As clinical flow cytometry practices continue to expand and immunophenotyping for leukemia and lymphoma becomes more widespread, the need for defined guidelines for training of medical professionals is imperative. Standards of expected knowledge and skills are necessary to ensure reliable test results as well as provide direction to those who are considering adding flow cytometry to their clinical laboratory practice. Before now, no clear guidelines have been established for defining the areas of responsibility, education and training standards, and credentials that would be required to perform clinical flow cytometry for leukemia and lymphoma. As part of the 2006 Bethesda Consensus conference, a committee was formed to address this need and provide recommendations for training and education. The committee included laboratory professionals from private, public, and university hospitals as well as large reference laboratories that routinely operate clinical flow cytometry laboratories with an emphasis on lymphoma and leukemia immunophenotyping. This document represents the work of the committee. Categories of work responsibility are defined and the requisite education, training, and credentials, as well as measurement methods for assessing competency for each area of responsibility are provided. Additional recommendations are included that promote creating a specialty certification in flow cytometry, establishing benchmarks for training technologists and interpreters, and offer suggestions for minimum levels of experience to direct a clinical flow cytometry laboratory.

2006 Bethesda International Consensus recommendations on the immunophenotypic analysis of hematolymphoid neoplasia by flow cytometry: optimal reagents and reporting for the flow cytometric diagnosis of hematopoietic neoplasia.

Immunophenotyping by flow cytometry has become standard practice in the evaluation and monitoring of patients with hematopoietic neoplasia. However, despite its widespread use, considerable variability continues to exist in the reagents used for evaluation and the format in which results are reported. As part of the 2006 Bethesda Consensus conference, a committee was formed to attempt to define a consensus set of reagents suitable for general use in the diagnosis and monitoring of hematopoietic neoplasms. The committee included laboratory professionals from private, public, and university hospitals as well as large reference laboratories that routinely operate clinical flow cytometry laboratories with an emphasis on lymphoma and leukemia immunophenotyping. A survey of participants successfully identified the cell lineage(s) to be evaluated for each of a variety of specific medical indications and defined a set of consensus reagents suitable for the initial evaluation of each cell lineage. Elements to be included in the reporting of clinical flow cytometric results for leukemia and lymphoma evaluation were also refined and are comprehensively listed. The 2006 Bethesda Consensus conference represents the first successful attempt to define a set of consensus reagents suitable for the initial evaluation of hematopoietic neoplasia.

Quality control in clinical flow cytometry.

Immunophenotyping by flow cytometry is a robust and highly complex technology used in the enumeration and characterization of leukocytes, including normal lymphocyte subsets and hematologic neoplasms. Samples consist of peripheral blood and many times irreplaceable samples, such as bone marrow and fresh tissue. These samples are collected, transported, prepared, analyzed, and interpreted, resulting in diagnostic and prognostic information. Such information is critical to treatment decisions for patients. In order to obtain accurate and reproducible results, it is essential to have optimized and standardized procedures, rigorous quality control, and assurance programs encompassing preanalytic, analytic, and postanalytic processes.

More than just quality control.

Providing quality flow cytometric results requires more than monitoring quality control data. Laboratories should standardize all aspects of testing and evaluate each one critically for opportunities to improve. This article discusses a complete quality management system that includes assay validation and change control, specimen collection and delivery, ordering of flow cytometric testing, sample preparation, verification of specimen integrity, flow cytometry data acquisition, analysis and interpretation, reporting of results, document of standard operating procedures, proficiency testing, training, and documentation of ongoing competency.

Flow cytometry quality requirements for monitoring of minimal disease in plasma cell myeloma.

Current therapeutic approaches for plasma cell myeloma (PCM) attain an overall survival of more than 6 years for the majority of newly diagnosed patients. However, PFS and OS are the only accepted FDA clinical endpoints for demonstrating drug efficacy before they can be become frontline therapeutic options. There is, however, recognition that the increasing gap between drug development and approval for mainstream therapeutic use needs to be shortened. As such regulatory bodies such as the FDA are now considering whether biomarker response evaluation, as in measurement of minimal residual disease (MRD) as assessed by flow cytometry (FC), can provide an early, robust prediction of survival and therefore improve the drug approval process. Recently, FC MRD using a standardized eight-color antibody methodology has been shown to have a minimum sensitivity of 0.01% and an upper sensitivity of 0.001%. To ensure that all laboratories using this approach achieve the same levels of sensitivity it is crucially important to have standardized quality management procedures in place. This manuscript accompanies those published in this special issue and describes the minimum that is required for validating and quality monitoring of this highly specific test to ensure any laboratory, irrespective of location, will achieve the expected quality standards required.

Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS - part I - rationale and aims.

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called "home brew" assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part I - Rationale and aims. © 2013 International Clinical Cytometry Society.

Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS - part IV - postanalytic considerations.

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called home brew assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part IV - Postanalytic considerations.