- Email: [email protected]
Recommendations for the evaluation of specimen stability for flow cytometric testing during drug development
Journal of Immunological Methods 418 (2015) 1–8
Contents lists available at ScienceDirect
Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim
Review
Recommendations for the evaluation of specimen stability for flow cytometric testing during drug development Lynette Brown a, Cherie L. Green b, Nicholas Jones c, Jennifer J. Stewart a, Stephanie Fraser d, Kathy Howell k, Yuanxin Xu e, Carla G. Hill f, Christopher A. Wiwi g, Wendy I. White h, Peter J. O'Brien i, Virginia Litwin j,⁎ a
Flow Contract Site Laboratory, LLC, 13029 NE 126th PL, Unit A229, Kirkland, WA 98034, USA Amgen, Inc., 1 Amgen Center Drive, Mailstop 30E-3-C, Thousand Oaks, CA 91320, USA c LabCorp Clinical Trials, Laboratory Corporation of America® Holdings, 201 Summit View Dr., Suite 200, Brentwood, TN 37027, USA d Pfizer, Eastern Point Rd., Groton, CT 06340, USA e Clinical Laboratory Sciences, DSAR, Sanofi, One The Mountain Road, Framingham, MA 01701, USA f 16 Rolling Lane, Hamilton, NJ 08690, USA g Celgene Cellular Therapeutics, 7 Powder Horn Drive, Warren, NJ 07059, USA h Medimmune, LLC, One MedImmune Way, Gaithersburg, MD 20878, USA i Pfizer Worldwide Research and Development, 10724 Science Center Drive, San Diego, CA 92121, USA j Covance Central Laboratory Services, 8211 SciCor Dr, Indianapolis, IN 46214, USA k 411 Walnut St., #7166, Green Cove Springs, FL 32043, USA b
a r t i c l e
i n f o
a b s t r a c t The objective of this manuscript is to present an approach for evaluating specimen stability for flow cytometric methods used during drug development. While this approach specifically addresses stability assessment for assays to be used in clinical trials with centralized testing facilities, the concepts can be applied to any stability assessment for flow cytometric methods. The proposed approach is implemented during assay development and optimization, and includes suggestions for designing a stability assessment plan, data evaluation and acceptance criteria. Given that no single solution will be applicable in all scenarios, this manuscript offers the reader a roadmap for stability assessment and is intended to guide the investigator during both the method development phase and in the experimental design of the validation plan. © 2015 Elsevier B.V. All rights reserved.
Article history: Received 15 July 2014 Received in revised form 18 October 2014 Accepted 24 January 2015 Available online 4 February 2015 Keywords: Flow cytometry Specimen stability Anticoagulants Cell stabilization Biomarker Clinical trials
Contents 1. 2.
Introduction . . . . . . . . . . . Specimen stability assessment . . . 2.1. Process overview . . . . . . 2.2. Specimen type and collection 2.2.1. EDTA . . . . . . . 2.2.2. Sodium heparin . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
⁎ Corresponding author. E-mail addresses: [email protected] (L. Brown), [email protected] (C.L. Green), [email protected] (N. Jones), [email protected] (J.J. Stewart), Stephanie.Fraser@pfizer.com (S. Fraser), [email protected] (K. Howell), [email protected] (Y. Xu), [email protected] (C.G. Hill), [email protected] (C.A. Wiwi), [email protected] (W.I. White), Peter.OBrien2@pfizer.com (P.J. O'Brien), [email protected] (V. Litwin).
http://dx.doi.org/10.1016/j.jim.2015.01.008 0022-1759/© 2015 Elsevier B.V. All rights reserved.
. . . . . .
2 2 2 2 2 3
2
L. Brown et al. / Journal of Immunological Methods 418 (2015) 1–8
2.2.3. Citrate anticoagulants . . . . . . . . . . 2.2.4. Stabilization tubes . . . . . . . . . . . 2.3. Post-collection considerations . . . . . . . . . . 2.3.1. Peripheral blood mononuclear cells (PBMC) 2.3.2. Post-processing stability . . . . . . . . . 3. Experimental design . . . . . . . . . . . . . . . . . . 4. Data evaluation . . . . . . . . . . . . . . . . . . . . 4.1. Visual inspection . . . . . . . . . . . . . . . . 4.2. Statistical evaluation . . . . . . . . . . . . . . . 5. Acceptance criteria . . . . . . . . . . . . . . . . . . . 6. Summary . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Multiparametric flow cytometry is the leading technology for the simultaneous characterization of individual cells. When suitable reagents are available, flow cytometry can be used to determine the developmental phenotype and functional status of a cell, including its activation state, developmental stage, cell cycle status, and signal transduction pathway engagement. Flow cytometry is useful across all phases of drug development. Examples include analyses of drug target occupancy and pharmacodynamics in pre-clinical and clinical studies, as well as determination of patient eligibility and stratification for clinical trials, and assessment of study endpoints (Green et al., 2011; O'Hara et al., 2011). Typically, later stage drug development clinical trials include multiple investigative sites distributed globally, necessitating the need for specimens to be shipped to a centralized testing facility. The advantages of centralized analysis are a significant decrease in the variability associated with differences in sample processing, instrumentation, and data analysis. The primary challenge associated with centralized testing is the delay in testing after specimen collection. Thus, a thorough assessment of specimen stability is critical to successful centralized testing. In this manuscript we propose a process for assessing specimen stability, with an emphasis on the challenges associated with specimen stability in cell-based fluorescence methods. 2. Specimen stability assessment Key variables that affect stability include specimen type, sample collection methods, and assay design such as monoclonal antibody (mAb) clone selection, and fluorochrome/antigen pairing. Logistical considerations such as transportation temperature and time also impact specimen stability. Considerations regarding specimen stability should be incorporated into the assay development process (Fig. 1). If specimen stability does not meet the requirements for the intended use of the assay, reconfiguring the assay (e.g. new anticoagulant, mAb clones, staining conditions) may result in increased stability. 2.1. Process overview After initial determination of the assay objective, the flow cytometry panel is designed and the type of specimen and collection procedure established. Next, the assay should be
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
4 4 4 4 4 4 5 5 6 6 6 7 7
fully optimized with regard to antibody titration, staining conditions (time and temperature), lyse, wash and fixation sequences and buffer selection (Tanqri et al., 2013). Method validation and stability assessment should begin only after the assay has been fully optimized. Initial stability assessments should be conducted with specimens stored in the laboratory at ambient temperature (18 to 26 °C); later the stability of shipped samples should also be evaluated. When assessing the stability of shipped samples, it is important to consider geographic locations and seasonal temperature fluctuations that may be encountered within the clinical study. If acceptable stability is not achieved with specimens maintained at ambient temperature, storage at 2 to 8 °C should be considered. Refrigeration may preserve specimen stability but may also increase the risk of clotting (CLSI, 2007) and alter surface antigen expression. For temperature sensitive assays, insulated shipping containers and refrigerants such as gel packs may be required to maintain the required temperature during transit. In this case, temperature tracking devices are recommended as they provide additional quality monitoring data. 2.2. Specimen type and collection For clinical trials, peripheral whole blood is the most frequently collected specimen type for flow cytometric analysis. Bone marrow, cerebral spinal fluid, synovial fluid, and tissue biopsies require more invasive collection techniques and are used less often as a result. Whole blood is typically drawn by venipuncture into vacuum tubes containing anticoagulant, and in some cases, a preservative or stabilization solution. The choice of anticoagulant and blood collection tube is often driven by logistical considerations and the type of assay being performed (e.g., immunophenotyping, and assessments of signal transduction and other intracellular functions). In all cases, a thorough understanding of exactly what will be measured and the intended use of the data, is required for appropriate anticoagulant selection (Narayanan, 2000). Each anticoagulant has potential advantages and limitations as discussed below (Carter et al., 1992; Son et al., 1996; McCarthy, 2007). 2.2.1. EDTA Ethylenediaminetetraacetic acid (EDTA) is available in several different formulations, has several distinct advantages
L. Brown et al. / Journal of Immunological Methods 418 (2015) 1–8
3
Assay Development
Stability Assessment Process
Assay objective (immunophenotyping, intracellular cytokines, phoso-proteins)
Re-develop assay (new anticoagulant, mAb clones, staining conditions)
Evaluate anticoagulant
Assay optimization
Assay samples at baseline and at various time points NO
Stability Assessment
Is sample stable for at least 24 hours at the testing bench?
YES
Shipped sample stability demonstrated?
NO
Consider temperature monitors, shipping temperatures, stabilizers, etc.
YES
Evaluate post-processing stability
Proceed with assay within constraints of stability
Fig. 1. Stability assessment process: overview of the recommended process for evaluating specimen stability for flow cytometric methods. This includes determining the assay objective and the type of specimen required. The assay is then fully optimized and stability assessed. In some cases, stability is assessed as a consideration in optimizing the assay, so the Assay Development/Stability Assessment is combined.
over other anticoagulants, and is commonly used for flow cytometry. Blood collected in EDTA maintains most cell surface antigens and is preferred when flow cytometric results will be correlated with hematology analyzer data for the calculation of absolute cell counts. Several of the flow cytometric in vitro diagnostic assays require EDTA (BD, 2011; Coulter, 2012). Although some lymphocyte cell surface markers have been reported to be stable in EDTA for up to 72 h after collection, changes in the myeloid and monocytic populations can often restrict overall specimen stability to less than 48 h (Stelzer et al., 1997; Bergeron et al., 2002; Mandy et al., 2003; Olteanu et al., 2012). EDTA chelates divalent cations and thus it is not
suitable in assays measuring calcium dependent interaction or in ex vivo activation (Repo et al., 1995; Jayachandran et al., 2012). Moreover, the epitopes of certain monoclonal antibodies are Ca++ or Mg++ dependent and thus the use of EDTA may negatively impact the binding of the detecting antibodies. 2.2.2. Sodium heparin Sodium heparin is a suitable anticoagulant for cell surface immunophenotyping and functional assays such as intracellular cytokines, internalization, and cell signaling. For many assays, heparinized blood can maintain surface marker integrity and cellular function for several days (Mandy et al., 2003).
4
L. Brown et al. / Journal of Immunological Methods 418 (2015) 1–8
Cells shipped at ambient temperature or higher (such as 37 °C) are metabolically active and need Mg2+ and Ca2+ for their viability. In contrast to blood collected in EDTA, heparinized blood preserves these ions and maintains cell viability and functionality for longer period of time during shipping. Lyophilized formulations that minimize dilution, a critical consideration in the direct assessment of absolute cell counts, are available. 2.2.3. Citrate anticoagulants Acid Citrate Dextrose (ACD) and Sodium Citrate are anticoagulants reported to maintain the integrity and function of platelets in whole blood, and are therefore recommended for flow cytometric assays involving platelet enumeration or activation (Kuhne et al., 1995; Robert et al., 2009). Citratebased blood collection tubes contain liquid formulations and thus present challenges in the direct assessment of absolute cell counts (Mandy et al., 2003). Moreover, underfilled tubes will result in concentrations of ACD which may be toxic to cells (von Pape et al., 2000). 2.2.4. Stabilization tubes Blood stabilization tubes (e.g., Cyto-Chex® BCT (Streck, Inc.), TransFix® (Affiniti, LLC), and CellSave™ (Immunivest Corporation)) provide anticoagulation with proprietary stabilization compounds purported to extended specimen stability. Stability of light scatter and some surface markers have been reported for up to 14 days post-collection (Warrino et al., 2005; Plate et al., 2009; Davis et al., 2011; Ng et al., 2012) These products contain fixatives which render the samples unacceptable for functional studies. Furthermore, the fixatives may denature certain epitopes resulting in decreased binding by certain mAb. Thus it is prudent to compare blood stabilization tubes to anticoagulated blood to verify antigenic integrity. Lastly, these products contain liquid anticoagulants which require that the tubes are fully filled to avoid adverse effects. 2.3. Post-collection considerations 2.3.1. Peripheral blood mononuclear cells (PBMC) In some circumstances, the assay matrix will be peripheral blood mononuclear cells (PBMC) rather than whole blood. Depending on the stability of the antigens and cellular populations, PBMC isolation may be performed on shipped whole blood or if stability cannot be established, at the site of collection. Stability assessments from time of collection to PBMC isolation and cryopreservation should be evaluated (Mallone et al., 2011; Olson et al., 2011) (Fig. 2). If PBMC are to be cryopreserved, the assessment of stability immediately following PBMC isolation and prior to cryopreservation is necessary to generate a baseline so that the impact of storage can be adequately evaluated (Bull et al., 2007). Ideally, frozen specimen stability should be evaluated for the duration of the anticipated storage time. If this is not possible, ongoing stability studies should be incorporated into the study design such that designated “stability control samples” may be run in parallel with test specimens over the duration of the study. Parameters to evaluate related to PBMC stability can include cell viability, yield, and purity, but must also include a
consideration of the final downstream application (Fowke et al., 2000; Lee et al., 2006; Chau et al., 2008). 2.3.2. Post-processing stability The stability of the processed samples (stained/fixed) should also be evaluated so that the processed sample does not degrade prior to acquisition on the instrument. Unfixed cells can change over time, antibodies can be internalized with increasing temperatures, and fluorophores can degrade when exposed to environmental factors. Time, temperature, and exposure to light will affect each fluorophore differently and may lead to decreased processed sample stability. Ideally, samples should be analyzed within 30 min of staining, but when this is not feasible, processed sample stability must be determined (Cunliffe et al., 2009). 3. Experimental design The International Standards Organization (ISO) has defined sample stability as the capability of a sample material to retain the initial property of a measured constituent for a period of time within specified limits when the sample is stored under defined conditions (ISO Guide 30/92-2.7). Specimen stability assessments are designed to monitor changes in sample integrity over time to determine acceptable limits for sample use in a given assay. Key to this process is the identification of differences in data obtained from specimens analyzed immediately after collection and at relevant post-collection intervals. For each stability assessment, a validation plan should be prepared in which the number of samples, number of replicates, time points, a description of the baseline samples, and acceptance criteria are defined. The type of assay and intended use of the data will influence the experimental design. A minimum of five apparently healthy subjects has been recommended for stability assessment (Wood et al., 2013). The value of assessing disease state samples for stability has been called into question as there are no convincing studies indicating that the stability of those specimens would differ from non-diseased (Wood et al., 2013). However, some circumstances may warrant incorporation of relevant disease samples. In scenarios, such as leukemia and lymphomas, where the target cell population is not present in healthy subjects the use of disease state samples is of value. Considerations regarding the time from specimen collection to testing should influence the time points selected in the stability evaluation. It is a good practice to include at least one time point beyond the anticipated optimal sample processing time in the stability assessment or, if feasible, to extend the evaluation until decreased stability is observed. Defining the timepoint for the baseline sample is not always straightforward and, as always, considerations regarding the assay objective, intended use of the data, and shipment time after specimen collection site must be taken into consideration. Ideally, the specimen should be analyzed immediately after collection. For some enzymatic assays and functional assays, the baseline sample may need to be processed or stabilized within 2 h. If fresh specimens are not available, the assay baseline may be established using the earliest practical time after specimen delivery. In this scenario,
L. Brown et al. / Journal of Immunological Methods 418 (2015) 1–8
5
PBMC Stability Assessment
Pre-PBMC Isolation Condition
Effect of Whole Blood Storage and Shipping
Evaluate stability of whole blood from collection through PBMC preparation
Determine effect of whole blood shipment
Is sample stable after shipping?
NO
Process PBMC at collection site
NO
Assay freshly isolated PBMC
YES
Proceed with assay within the constraints of stability
Post-PBMC Isolation
Effect of PBMC Cryopreservation
If applicable, determine effect of frozen PBMC storage
Acceptable recoveries and performance in the final assay for frozen PBMC storage conditions? YES
Proceed with assay within the constraints of stability
If possible, perform routine stability assessment on cryopreserved samples.
Fig. 2. PBMC stability assessment: steps for evaluating specimen stability for flow cytometric methods that are specific to PBMC specimens. Stability evaluation should include pre- and post-isolation assessments of the PBMC specimen. The stability of the target analyte may be influenced from collection through PBMC preparation to the method of cryopreservation, therefore, all steps should be given careful consideration during assay development.
the baseline specimen may be about 24 h post-collection. The caveat to this approach is that changes that occur within the first 24 h after collection are not fully assessed in the stability exercise. Therefore, every effort should be made to obtain a fresh baseline specimen as this will provide the best assessment of specimen degradation. If specimens are found to be unstable at 24 h or 48 h, even after reconfiguring the assay design, stability testing at earlier time points for example, at six or twelve hour intervals may need to be evaluated in order to determine the stability limits. For specimens with less that 24–48 hour stability the use of centralized testing laboratories becomes logistically more challenging.
4. Data evaluation Data evaluation must include both statistical analyses of changes from baseline conditions as well as visual examination of the specimen and the flow cytometric data.
4.1. Visual inspection Pre-analytical considerations start with visual inspection and an assessment of the specimen prior to proceeding with the analytical method. Inclusion of rejection criteria such as evidence of clotting or hemolysis is the first step in ensuring
6
L. Brown et al. / Journal of Immunological Methods 418 (2015) 1–8
5. Acceptance criteria
acceptable specimen quality. Data analysis include assessment of changes in light scatter and antigen properties, loss of viability or membrane integrity, and changes in cellular composition or counts (Fig. 3). Alterations in these properties can accelerate with the age of the specimen A (Elghetany and Davis, 2005). Some of these issues may result in an immediate rejection of the specimen whereas other observations may warrant interpretation with caution or repeat analysis (Table 1).
In establishing the acceptance criteria, especially in drug development, the intended use of the data must be considered (O'Hara et al., 2011). Will the results be used as an exploratory biomarker, an efficacy endpoint or enrollment criteria? What types of changes are expected? Less than a 20% difference between the baseline specimen value and the stored specimen value is the most commonly used acceptance criteria. In certain cases, such as rare event detection or analysis of antigen expression a higher percent change may be acceptable. Moreover, in assays with high imprecision, the acceptance criteria for stability must also be in alignment with the assay precision (DeSilva et al., 2003; Donnenberg and Donnenberg, 2007; Maecker et al., 2008). The comparison of results obtained from a stored specimen and the baseline specimen may be within the inter-assay precision, yet may exceed a 20% change from baseline. Conversely, for an assay with exquisite precision, a 20% difference may be unacceptable. Specimen stability can thus be established at the latest time point where a minimum of 80% of the validation samples meet the acceptance criteria (Wood et al., 2013).
4.2. Statistical evaluation The most common descriptive statistic for stability assessment is the percent change calculated using one of the following formula (Belouski et al., 2010): ½ðStability time point – BaselineÞ Baseline 100 or ½1 – ðStability time point BaselineÞ 100:
6. Summary All methods require appropriate specimen stability evaluation to ensure that reliable data are obtained. Factors influencing
It is also useful to determine the coefficient of variation (CV) between the baseline value and each time point.
24 hours post collection
48 hours post collection
SODIUM HEPARIN
SSC
EDTA
< 2 hours post collection
FSC Fig. 3. Assessment of light scatter degradation over time. Light scatter (forward scatter vs. side scatter) properties in whole blood specimens from a healthy donor collected in EDTA (top panel) and Na heparin (bottom panel) tubes and stored at ambient temperature for up to 48 h post collection. Changes in light scatter properties of the granulocyte compartment are evident in EDTA blood at 24 h post draw. Data was generated at Amgen, Inc., Thousand Oaks, Ca.
L. Brown et al. / Journal of Immunological Methods 418 (2015) 1–8
7
Table 1 Visual inspection guide. Stage
Parameters
Observation method
Impact
Action
Pre-analytical
Hemolysis
Visual inspection of blood specimen
Establish criteria for specimen rejection (e.g. hemolysis observed = reject)
Pre analytical
Clotting/cell aggregation
Visual inspection of blood specimen
Extreme storage conditions may result in excessive RBC lysis during transport resulting in comprised specimen integrity Inability to accurately characterize immune compartment due to selective loss or alterations in subpopulations
Pre-analytical
Partial draw
Visual inspection of blood specimen
Post-analytical
Light scatter changes
Visual inspection of forward and side scatter plot
Incomplete blood draw may result in increased concentration of anticoagulant or preservative resulting in hypertonic conditions with potentially adverse effects Storage time and temperature may adversely impact cell populations with cytoplasmic granules (e.g. granulocytes), degranulation results in decreased side scatter
specimen stability for flow cytometric assays include the assay format, the choice of anticoagulant or cell stabilization reagent, post-collection specimen processing, temperature, and shipping conditions. The approach suggested within this manuscript is intended to serve as a framework for the evaluation of sample stability as it relates to utilizing a central facility for processing and analysis. As each assay application is unique; it is important to structure the evaluation of stability in a manner that is appropriate for the intended use of the assay. If a given parameter does not meet the stability acceptance criteria, increased stability may be achieved by re-optimizing the assay design (e.g. clone selection, fluorochrome assignment, gating strategy).
Acknowledgments The authors thank the members of the American Association of Pharmaceutical Scientists for review of the manuscript. The assistance of Sarah Livingston in preparing the flow charts is gratefully acknowledged.
References BD Biosciences, 2011. BD multitest™ 6-color TBNK reagent with Trucount tubes. Product Number 23-10834-02. BD Biosciences, San Jose, CA (http:// www.bdbiosciences.com). Beckman Coulter, 2012. CYTO-STAT® tetraCHROME™ CD45-FITC/CD4-RD1/ CD8-ECD/CD3-PC5 and CYTO-STAT® tetraCHROME™ CD45-FITC/CD56RD1/CD19-ECD/CD3-PC5. Product Number 4238068-KE. Beckman Coulter, Brea, CA (http://www.beckmancoulter.com). Belouski, S.S., Wallace, D., Weisman, M., Ishimori, M., Hendricks, L., Zack, D., Vincent, M., Rasmussen, E., Ferbas, J., Chung, J., 2010. Sample stability and variability of B-cell subsets in blood from healthy subjects and patients with systemic lupus erythematosus. Cytometry B Clin. Cytom. 78, 49. Bergeron, M., Nicholson, J.K., Phaneuf, S., Ding, T., Soucy, N., Badley, A.D., Hawley Foss, N.C., Mandy, F., 2002. Selection of lymphocyte gating protocol has an impact on the level of reliability of T-cell subsets in aging specimens. Cytometry 50, 53. Bull, M., Lee, D., Stucky, J., Chiu, Y.L., Rubin, A., Horton, H., McElrath, M.J., 2007. Defining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for HIV vaccine trials. J. Immunol. Methods 322, 57. Carter, P.H., Resto-Ruiz, S., Washington, G.C., Ethridge, S., Palini, A., Vogt, R., Waxdal, M., Fleisher, T., Noguchi, P.D., Marti, G.E., 1992. Flow cytometric
Establish criteria for specimen rejection (e.g. clots are evident = cancelation or clots are evident = proceed with caution and monitor frequency of doublets in light scatter area vs. height plot) Establish criteria for specimen rejection (e.g. bhalf expected volume = reject)
Establish criteria for specimen rejection
analysis of whole blood lysis, three anticoagulants, and five cell preparations. Cytometry 13, 68. Chau, C.H., Rixe, O., McLeod, H., Figg, W.D., 2008. Validation of analytic methods for biomarkers used in drug development. Clin. Cancer Res. 14, 5967. CLSI, 2007. Enumeration of immunologically defined cell populations by flow cytometry; approve guideline H42-A2. Clinical Laboratory Standards Institute vol. 27. Clinical Laboratory Standards Institute. Cunliffe, J., Derbyshire, N., Keeler, S., Coldwell, R., 2009. An approach to the validation of flow cytometry methods. Pharm. Res. 26, 2551. Davis, C., Wu, X., Li, W., Fan, H., Reddy, M., 2011. Stability of immunophenotypic markers in fixed peripheral blood for extended analysis using flow cytometry. J. Immunol. Methods 363, 158. DeSilva, B., Smith, W., Weiner, R., Kelley, M., Smolec, J., Lee, B., Khan, M., Tacey, R., Hill, H., Celniker, A., 2003. Recommendations for the bioanalytical method validation of ligand-binding assays to support pharmacokinetic assessments of macromolecules. Pharm. Res. 20, 1885. Donnenberg, A.D., Donnenberg, V.S., 2007. Rare-event analysis in flow cytometry. Clin. Lab. Med. 27, 627 (viii). Elghetany, M.T., Davis, B.H., 2005. Impact of preanalytical variables on granulocytic surface antigen expression: a review. Cytometry B Clin. Cytom. 65, 1. Fowke, K.R., Behnke, J., Hanson, C., Shea, K., Cosentino, L.M., 2000. Apoptosis: a method for evaluating the cryopreservation of whole blood and peripheral blood mononuclear cells. J. Immunol. Methods 244, 139. Green, C.L., Brown, L., Stewart, J.J., Xu, Y., Litwin, V., Mc Closkey, T.W., 2011. Recommendations for the validation of flow cytometric testing during drug development: I instrumentation. J. Immunol. Methods 363, 104. ISO Guide 30/92-2.7. In: Terms and definitions used in connection with reference materials. Jayachandran, M., Miller, V.M., Heit, J.A., Owen, W.G., 2012. Methodology for isolation, identification and characterization of microvesicles in peripheral blood. J. Immunol. Methods 375, 207. Kuhne, T., Hornstein, A., Semple, J., Chang, W., Blanchette, V., Freedman, J., 1995. Flow cytometric evaluation of platelet activation in blood collected into EDTA vs. Diatube-H, a sodium citrate solution supplemented with theophylline, adenosine, and dipyridamole. Am. J. Hematol. 50, 40–45. Lee, J.W., Devanarayan, V., Barrett, Y.C., Weiner, R., Allinson, J., Fountain, S., Keller, S., Weinryb, I., Green, M., Duan, L., Rogers, J.A., Millham, R., O'Brien, P.J., Sailstad, J., Khan, M., Ray, C., Wagner, J.A., 2006. Fit-for-purpose method development and validation for successful biomarker measurement. Pharm. Res. 23, 312. Maecker, H.T., Hassler, J., Payne, J.K., Summers, A., Comatas, K., Ghanayem, M., Morse, M.A., Clay, T.M., Lyerly, H.K., Bhatia, S., Ghanekar, S.A., Maino, V.C., Delarosa, C., Disis, M.L., 2008. Precision and linearity targets for validation of an IFNgamma ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides. BMC Immunol. 9, 9. Mallone, R., Mannering, S.I., Brooks-Worrell, B.M., Durinovic-Bello, I., Cilio, C.M., Wong, F.S., Schloot, N.C., T-Cell Workshop Committee, I.o.D.S., 2011. Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society. Clin. Exp. Immunol. 163, 33.
8
L. Brown et al. / Journal of Immunological Methods 418 (2015) 1–8
Mandy, F.F., Nicholson, J.K., McDougal, J.S., Cdc, 2003. Guidelines for performing single-platform absolute CD4+ T-cell determinations with CD45 gating for persons infected with human immunodeficiency virus. Centers for Disease Control and Prevention. MMWR. Recommendations and reports: morbidity and mortality weekly report. Recommendations and Reports/Centers for Disease Control 52, p. 1. McCarthy, D.A., 2007. Flow Cytometry: Principles and Applications. In: Macey, M.G. (Ed.), Humana Press Inc, Totowa, NJ. Narayanan, S., 2000. The preanalytic phase. An important component of laboratory medicine. Am. J. Clin. Pathol. 113, 429. Ng, A.A., Lee, B.T., Teo, T.S., Poidinger, M., Connolly, J.E., 2012. Optimal cellular preservation for high dimensional flow cytometric analysis of multicentre trials. J. Immunol. Methods 385, 79. O'Hara, D.M., Xu, Y., Liang, Z., Reddy, M.P., Wu, D.Y., Litwin, V., 2011. Recommendations for the validation of flow cytometric testing during drug development: II assays. J. Immunol. Methods 363, 120. Olson, W.C., Smolkin, M.E., Farris, E.M., Fink, R.J., Czarkowski, A.R., Fink, J.H., Chianese-Bullock, K.A., Slingluff Jr., C.L., 2011. Shipping blood to a central laboratory in multicenter clinical trials: effect of ambient temperature on specimen temperature, and effects of temperature on mononuclear cell yield, viability and immunologic function. J. Transl. Med. 9, 26. Olteanu, H., Schur, B.C., Harrington, A.M., Kroft, S.H., 2012. Time and temperature stability of T-cell subsets evaluated by a dual-platform method. Am. J. Blood Res. 2, 128. Plate, M.M., Louzao, R., Steele, P.M., Greengrass, V., Morris, L.M., Lewis, J., Barnett, D., Warrino, D., Hearps, A.C., Denny, T., Crowe, S.M., 2009. Evaluation of the blood stabilizers TransFix and Cyto-Chex BCT for lowcost CD4 T-cell methodologies. Viral Immunol. 22, 329. Repo, H., Jansson, S.E., Leirisalo-Repo, M., 1995. Anticoagulant selection influences flow cytometric determination of CD11b upregulation in vivo and ex vivo. J. Immunol. Methods 185, 65.
Robert, S., Poncelet, P., Lacroix, R., Arnaud, L., Giraudo, L., Hauchard, A., Sampol, J., Dignat-George, F., 2009. Standardization of platelet-derived microparticle counting using calibrated beads and a Cytomics FC500 routine flow cytometer: a first step towards multicenter studies? J. Thromb. Haemost. (JTH) 7, 190–197. Son, B.K., Roberts, R.L., Ank, B.J., Stiehm, E.R., 1996. Effects of anticoagulant, serum, and temperature on the natural killer activity of human peripheral blood mononuclear cells stored overnight. Clin. Diagn. Lab. Immunol. 3, 260. Stelzer, G.T., Marti, G., Hurley, A., McCoy Jr., P., Lovett, E.J., Schwartz, A., 1997. U.S.–Canadian consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: standardization and validation of laboratory procedures. Cytometry 30, 214. Tanqri, S., Vall, H., Kaplan, D., Hoffman, B., Purvis, N., Porwit, A., Hunsberger, B., Shankey, T.V., on behalf of, I.I.W.G., 2013. Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS – part III – analytical issues. Cytometry B Clin. Cytom. 84, 291. von Pape, K.W., Aland, E., Bohner, J., 2000. Platelet function analysis with PFA-100 in patients medicated with acetylsalicylic acid strongly depends on concentration of sodium citrate used for anticoagulation of blood sample. Thromb. Res. 98, 295. Warrino, D.E., DeGennaro, L.J., Hanson, M., Swindells, S., Pirruccello, S.J., Ryan, W.L., 2005. Stabilization of white blood cells and immunologic markers for extended analysis using flow cytometry. J. Immunol. Methods 305, 107. Wood, B., Jevremovic, D., Béné, M.C., Yan, M., Jacobs, P., Litwin, V., on behalf of, I.I.W.G., 2013. Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS – part V – assay performance criteria. Cytometry B Clin. Cytom. 84, 315.
Contents lists available at ScienceDirect
Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim
Review
Recommendations for the evaluation of specimen stability for flow cytometric testing during drug development Lynette Brown a, Cherie L. Green b, Nicholas Jones c, Jennifer J. Stewart a, Stephanie Fraser d, Kathy Howell k, Yuanxin Xu e, Carla G. Hill f, Christopher A. Wiwi g, Wendy I. White h, Peter J. O'Brien i, Virginia Litwin j,⁎ a
Flow Contract Site Laboratory, LLC, 13029 NE 126th PL, Unit A229, Kirkland, WA 98034, USA Amgen, Inc., 1 Amgen Center Drive, Mailstop 30E-3-C, Thousand Oaks, CA 91320, USA c LabCorp Clinical Trials, Laboratory Corporation of America® Holdings, 201 Summit View Dr., Suite 200, Brentwood, TN 37027, USA d Pfizer, Eastern Point Rd., Groton, CT 06340, USA e Clinical Laboratory Sciences, DSAR, Sanofi, One The Mountain Road, Framingham, MA 01701, USA f 16 Rolling Lane, Hamilton, NJ 08690, USA g Celgene Cellular Therapeutics, 7 Powder Horn Drive, Warren, NJ 07059, USA h Medimmune, LLC, One MedImmune Way, Gaithersburg, MD 20878, USA i Pfizer Worldwide Research and Development, 10724 Science Center Drive, San Diego, CA 92121, USA j Covance Central Laboratory Services, 8211 SciCor Dr, Indianapolis, IN 46214, USA k 411 Walnut St., #7166, Green Cove Springs, FL 32043, USA b
a r t i c l e
i n f o
a b s t r a c t The objective of this manuscript is to present an approach for evaluating specimen stability for flow cytometric methods used during drug development. While this approach specifically addresses stability assessment for assays to be used in clinical trials with centralized testing facilities, the concepts can be applied to any stability assessment for flow cytometric methods. The proposed approach is implemented during assay development and optimization, and includes suggestions for designing a stability assessment plan, data evaluation and acceptance criteria. Given that no single solution will be applicable in all scenarios, this manuscript offers the reader a roadmap for stability assessment and is intended to guide the investigator during both the method development phase and in the experimental design of the validation plan. © 2015 Elsevier B.V. All rights reserved.
Article history: Received 15 July 2014 Received in revised form 18 October 2014 Accepted 24 January 2015 Available online 4 February 2015 Keywords: Flow cytometry Specimen stability Anticoagulants Cell stabilization Biomarker Clinical trials
Contents 1. 2.
Introduction . . . . . . . . . . . Specimen stability assessment . . . 2.1. Process overview . . . . . . 2.2. Specimen type and collection 2.2.1. EDTA . . . . . . . 2.2.2. Sodium heparin . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
⁎ Corresponding author. E-mail addresses: [email protected] (L. Brown), [email protected] (C.L. Green), [email protected] (N. Jones), [email protected] (J.J. Stewart), Stephanie.Fraser@pfizer.com (S. Fraser), [email protected] (K. Howell), [email protected] (Y. Xu), [email protected] (C.G. Hill), [email protected] (C.A. Wiwi), [email protected] (W.I. White), Peter.OBrien2@pfizer.com (P.J. O'Brien), [email protected] (V. Litwin).
http://dx.doi.org/10.1016/j.jim.2015.01.008 0022-1759/© 2015 Elsevier B.V. All rights reserved.
. . . . . .
2 2 2 2 2 3
2
L. Brown et al. / Journal of Immunological Methods 418 (2015) 1–8
2.2.3. Citrate anticoagulants . . . . . . . . . . 2.2.4. Stabilization tubes . . . . . . . . . . . 2.3. Post-collection considerations . . . . . . . . . . 2.3.1. Peripheral blood mononuclear cells (PBMC) 2.3.2. Post-processing stability . . . . . . . . . 3. Experimental design . . . . . . . . . . . . . . . . . . 4. Data evaluation . . . . . . . . . . . . . . . . . . . . 4.1. Visual inspection . . . . . . . . . . . . . . . . 4.2. Statistical evaluation . . . . . . . . . . . . . . . 5. Acceptance criteria . . . . . . . . . . . . . . . . . . . 6. Summary . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Multiparametric flow cytometry is the leading technology for the simultaneous characterization of individual cells. When suitable reagents are available, flow cytometry can be used to determine the developmental phenotype and functional status of a cell, including its activation state, developmental stage, cell cycle status, and signal transduction pathway engagement. Flow cytometry is useful across all phases of drug development. Examples include analyses of drug target occupancy and pharmacodynamics in pre-clinical and clinical studies, as well as determination of patient eligibility and stratification for clinical trials, and assessment of study endpoints (Green et al., 2011; O'Hara et al., 2011). Typically, later stage drug development clinical trials include multiple investigative sites distributed globally, necessitating the need for specimens to be shipped to a centralized testing facility. The advantages of centralized analysis are a significant decrease in the variability associated with differences in sample processing, instrumentation, and data analysis. The primary challenge associated with centralized testing is the delay in testing after specimen collection. Thus, a thorough assessment of specimen stability is critical to successful centralized testing. In this manuscript we propose a process for assessing specimen stability, with an emphasis on the challenges associated with specimen stability in cell-based fluorescence methods. 2. Specimen stability assessment Key variables that affect stability include specimen type, sample collection methods, and assay design such as monoclonal antibody (mAb) clone selection, and fluorochrome/antigen pairing. Logistical considerations such as transportation temperature and time also impact specimen stability. Considerations regarding specimen stability should be incorporated into the assay development process (Fig. 1). If specimen stability does not meet the requirements for the intended use of the assay, reconfiguring the assay (e.g. new anticoagulant, mAb clones, staining conditions) may result in increased stability. 2.1. Process overview After initial determination of the assay objective, the flow cytometry panel is designed and the type of specimen and collection procedure established. Next, the assay should be
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
4 4 4 4 4 4 5 5 6 6 6 7 7
fully optimized with regard to antibody titration, staining conditions (time and temperature), lyse, wash and fixation sequences and buffer selection (Tanqri et al., 2013). Method validation and stability assessment should begin only after the assay has been fully optimized. Initial stability assessments should be conducted with specimens stored in the laboratory at ambient temperature (18 to 26 °C); later the stability of shipped samples should also be evaluated. When assessing the stability of shipped samples, it is important to consider geographic locations and seasonal temperature fluctuations that may be encountered within the clinical study. If acceptable stability is not achieved with specimens maintained at ambient temperature, storage at 2 to 8 °C should be considered. Refrigeration may preserve specimen stability but may also increase the risk of clotting (CLSI, 2007) and alter surface antigen expression. For temperature sensitive assays, insulated shipping containers and refrigerants such as gel packs may be required to maintain the required temperature during transit. In this case, temperature tracking devices are recommended as they provide additional quality monitoring data. 2.2. Specimen type and collection For clinical trials, peripheral whole blood is the most frequently collected specimen type for flow cytometric analysis. Bone marrow, cerebral spinal fluid, synovial fluid, and tissue biopsies require more invasive collection techniques and are used less often as a result. Whole blood is typically drawn by venipuncture into vacuum tubes containing anticoagulant, and in some cases, a preservative or stabilization solution. The choice of anticoagulant and blood collection tube is often driven by logistical considerations and the type of assay being performed (e.g., immunophenotyping, and assessments of signal transduction and other intracellular functions). In all cases, a thorough understanding of exactly what will be measured and the intended use of the data, is required for appropriate anticoagulant selection (Narayanan, 2000). Each anticoagulant has potential advantages and limitations as discussed below (Carter et al., 1992; Son et al., 1996; McCarthy, 2007). 2.2.1. EDTA Ethylenediaminetetraacetic acid (EDTA) is available in several different formulations, has several distinct advantages
L. Brown et al. / Journal of Immunological Methods 418 (2015) 1–8
3
Assay Development
Stability Assessment Process
Assay objective (immunophenotyping, intracellular cytokines, phoso-proteins)
Re-develop assay (new anticoagulant, mAb clones, staining conditions)
Evaluate anticoagulant
Assay optimization
Assay samples at baseline and at various time points NO
Stability Assessment
Is sample stable for at least 24 hours at the testing bench?
YES
Shipped sample stability demonstrated?
NO
Consider temperature monitors, shipping temperatures, stabilizers, etc.
YES
Evaluate post-processing stability
Proceed with assay within constraints of stability
Fig. 1. Stability assessment process: overview of the recommended process for evaluating specimen stability for flow cytometric methods. This includes determining the assay objective and the type of specimen required. The assay is then fully optimized and stability assessed. In some cases, stability is assessed as a consideration in optimizing the assay, so the Assay Development/Stability Assessment is combined.
over other anticoagulants, and is commonly used for flow cytometry. Blood collected in EDTA maintains most cell surface antigens and is preferred when flow cytometric results will be correlated with hematology analyzer data for the calculation of absolute cell counts. Several of the flow cytometric in vitro diagnostic assays require EDTA (BD, 2011; Coulter, 2012). Although some lymphocyte cell surface markers have been reported to be stable in EDTA for up to 72 h after collection, changes in the myeloid and monocytic populations can often restrict overall specimen stability to less than 48 h (Stelzer et al., 1997; Bergeron et al., 2002; Mandy et al., 2003; Olteanu et al., 2012). EDTA chelates divalent cations and thus it is not
suitable in assays measuring calcium dependent interaction or in ex vivo activation (Repo et al., 1995; Jayachandran et al., 2012). Moreover, the epitopes of certain monoclonal antibodies are Ca++ or Mg++ dependent and thus the use of EDTA may negatively impact the binding of the detecting antibodies. 2.2.2. Sodium heparin Sodium heparin is a suitable anticoagulant for cell surface immunophenotyping and functional assays such as intracellular cytokines, internalization, and cell signaling. For many assays, heparinized blood can maintain surface marker integrity and cellular function for several days (Mandy et al., 2003).
4
L. Brown et al. / Journal of Immunological Methods 418 (2015) 1–8
Cells shipped at ambient temperature or higher (such as 37 °C) are metabolically active and need Mg2+ and Ca2+ for their viability. In contrast to blood collected in EDTA, heparinized blood preserves these ions and maintains cell viability and functionality for longer period of time during shipping. Lyophilized formulations that minimize dilution, a critical consideration in the direct assessment of absolute cell counts, are available. 2.2.3. Citrate anticoagulants Acid Citrate Dextrose (ACD) and Sodium Citrate are anticoagulants reported to maintain the integrity and function of platelets in whole blood, and are therefore recommended for flow cytometric assays involving platelet enumeration or activation (Kuhne et al., 1995; Robert et al., 2009). Citratebased blood collection tubes contain liquid formulations and thus present challenges in the direct assessment of absolute cell counts (Mandy et al., 2003). Moreover, underfilled tubes will result in concentrations of ACD which may be toxic to cells (von Pape et al., 2000). 2.2.4. Stabilization tubes Blood stabilization tubes (e.g., Cyto-Chex® BCT (Streck, Inc.), TransFix® (Affiniti, LLC), and CellSave™ (Immunivest Corporation)) provide anticoagulation with proprietary stabilization compounds purported to extended specimen stability. Stability of light scatter and some surface markers have been reported for up to 14 days post-collection (Warrino et al., 2005; Plate et al., 2009; Davis et al., 2011; Ng et al., 2012) These products contain fixatives which render the samples unacceptable for functional studies. Furthermore, the fixatives may denature certain epitopes resulting in decreased binding by certain mAb. Thus it is prudent to compare blood stabilization tubes to anticoagulated blood to verify antigenic integrity. Lastly, these products contain liquid anticoagulants which require that the tubes are fully filled to avoid adverse effects. 2.3. Post-collection considerations 2.3.1. Peripheral blood mononuclear cells (PBMC) In some circumstances, the assay matrix will be peripheral blood mononuclear cells (PBMC) rather than whole blood. Depending on the stability of the antigens and cellular populations, PBMC isolation may be performed on shipped whole blood or if stability cannot be established, at the site of collection. Stability assessments from time of collection to PBMC isolation and cryopreservation should be evaluated (Mallone et al., 2011; Olson et al., 2011) (Fig. 2). If PBMC are to be cryopreserved, the assessment of stability immediately following PBMC isolation and prior to cryopreservation is necessary to generate a baseline so that the impact of storage can be adequately evaluated (Bull et al., 2007). Ideally, frozen specimen stability should be evaluated for the duration of the anticipated storage time. If this is not possible, ongoing stability studies should be incorporated into the study design such that designated “stability control samples” may be run in parallel with test specimens over the duration of the study. Parameters to evaluate related to PBMC stability can include cell viability, yield, and purity, but must also include a
consideration of the final downstream application (Fowke et al., 2000; Lee et al., 2006; Chau et al., 2008). 2.3.2. Post-processing stability The stability of the processed samples (stained/fixed) should also be evaluated so that the processed sample does not degrade prior to acquisition on the instrument. Unfixed cells can change over time, antibodies can be internalized with increasing temperatures, and fluorophores can degrade when exposed to environmental factors. Time, temperature, and exposure to light will affect each fluorophore differently and may lead to decreased processed sample stability. Ideally, samples should be analyzed within 30 min of staining, but when this is not feasible, processed sample stability must be determined (Cunliffe et al., 2009). 3. Experimental design The International Standards Organization (ISO) has defined sample stability as the capability of a sample material to retain the initial property of a measured constituent for a period of time within specified limits when the sample is stored under defined conditions (ISO Guide 30/92-2.7). Specimen stability assessments are designed to monitor changes in sample integrity over time to determine acceptable limits for sample use in a given assay. Key to this process is the identification of differences in data obtained from specimens analyzed immediately after collection and at relevant post-collection intervals. For each stability assessment, a validation plan should be prepared in which the number of samples, number of replicates, time points, a description of the baseline samples, and acceptance criteria are defined. The type of assay and intended use of the data will influence the experimental design. A minimum of five apparently healthy subjects has been recommended for stability assessment (Wood et al., 2013). The value of assessing disease state samples for stability has been called into question as there are no convincing studies indicating that the stability of those specimens would differ from non-diseased (Wood et al., 2013). However, some circumstances may warrant incorporation of relevant disease samples. In scenarios, such as leukemia and lymphomas, where the target cell population is not present in healthy subjects the use of disease state samples is of value. Considerations regarding the time from specimen collection to testing should influence the time points selected in the stability evaluation. It is a good practice to include at least one time point beyond the anticipated optimal sample processing time in the stability assessment or, if feasible, to extend the evaluation until decreased stability is observed. Defining the timepoint for the baseline sample is not always straightforward and, as always, considerations regarding the assay objective, intended use of the data, and shipment time after specimen collection site must be taken into consideration. Ideally, the specimen should be analyzed immediately after collection. For some enzymatic assays and functional assays, the baseline sample may need to be processed or stabilized within 2 h. If fresh specimens are not available, the assay baseline may be established using the earliest practical time after specimen delivery. In this scenario,
L. Brown et al. / Journal of Immunological Methods 418 (2015) 1–8
5
PBMC Stability Assessment
Pre-PBMC Isolation Condition
Effect of Whole Blood Storage and Shipping
Evaluate stability of whole blood from collection through PBMC preparation
Determine effect of whole blood shipment
Is sample stable after shipping?
NO
Process PBMC at collection site
NO
Assay freshly isolated PBMC
YES
Proceed with assay within the constraints of stability
Post-PBMC Isolation
Effect of PBMC Cryopreservation
If applicable, determine effect of frozen PBMC storage
Acceptable recoveries and performance in the final assay for frozen PBMC storage conditions? YES
Proceed with assay within the constraints of stability
If possible, perform routine stability assessment on cryopreserved samples.
Fig. 2. PBMC stability assessment: steps for evaluating specimen stability for flow cytometric methods that are specific to PBMC specimens. Stability evaluation should include pre- and post-isolation assessments of the PBMC specimen. The stability of the target analyte may be influenced from collection through PBMC preparation to the method of cryopreservation, therefore, all steps should be given careful consideration during assay development.
the baseline specimen may be about 24 h post-collection. The caveat to this approach is that changes that occur within the first 24 h after collection are not fully assessed in the stability exercise. Therefore, every effort should be made to obtain a fresh baseline specimen as this will provide the best assessment of specimen degradation. If specimens are found to be unstable at 24 h or 48 h, even after reconfiguring the assay design, stability testing at earlier time points for example, at six or twelve hour intervals may need to be evaluated in order to determine the stability limits. For specimens with less that 24–48 hour stability the use of centralized testing laboratories becomes logistically more challenging.
4. Data evaluation Data evaluation must include both statistical analyses of changes from baseline conditions as well as visual examination of the specimen and the flow cytometric data.
4.1. Visual inspection Pre-analytical considerations start with visual inspection and an assessment of the specimen prior to proceeding with the analytical method. Inclusion of rejection criteria such as evidence of clotting or hemolysis is the first step in ensuring
6
L. Brown et al. / Journal of Immunological Methods 418 (2015) 1–8
5. Acceptance criteria
acceptable specimen quality. Data analysis include assessment of changes in light scatter and antigen properties, loss of viability or membrane integrity, and changes in cellular composition or counts (Fig. 3). Alterations in these properties can accelerate with the age of the specimen A (Elghetany and Davis, 2005). Some of these issues may result in an immediate rejection of the specimen whereas other observations may warrant interpretation with caution or repeat analysis (Table 1).
In establishing the acceptance criteria, especially in drug development, the intended use of the data must be considered (O'Hara et al., 2011). Will the results be used as an exploratory biomarker, an efficacy endpoint or enrollment criteria? What types of changes are expected? Less than a 20% difference between the baseline specimen value and the stored specimen value is the most commonly used acceptance criteria. In certain cases, such as rare event detection or analysis of antigen expression a higher percent change may be acceptable. Moreover, in assays with high imprecision, the acceptance criteria for stability must also be in alignment with the assay precision (DeSilva et al., 2003; Donnenberg and Donnenberg, 2007; Maecker et al., 2008). The comparison of results obtained from a stored specimen and the baseline specimen may be within the inter-assay precision, yet may exceed a 20% change from baseline. Conversely, for an assay with exquisite precision, a 20% difference may be unacceptable. Specimen stability can thus be established at the latest time point where a minimum of 80% of the validation samples meet the acceptance criteria (Wood et al., 2013).
4.2. Statistical evaluation The most common descriptive statistic for stability assessment is the percent change calculated using one of the following formula (Belouski et al., 2010): ½ðStability time point – BaselineÞ Baseline 100 or ½1 – ðStability time point BaselineÞ 100:
6. Summary All methods require appropriate specimen stability evaluation to ensure that reliable data are obtained. Factors influencing
It is also useful to determine the coefficient of variation (CV) between the baseline value and each time point.
24 hours post collection
48 hours post collection
SODIUM HEPARIN
SSC
EDTA
< 2 hours post collection
FSC Fig. 3. Assessment of light scatter degradation over time. Light scatter (forward scatter vs. side scatter) properties in whole blood specimens from a healthy donor collected in EDTA (top panel) and Na heparin (bottom panel) tubes and stored at ambient temperature for up to 48 h post collection. Changes in light scatter properties of the granulocyte compartment are evident in EDTA blood at 24 h post draw. Data was generated at Amgen, Inc., Thousand Oaks, Ca.
L. Brown et al. / Journal of Immunological Methods 418 (2015) 1–8
7
Table 1 Visual inspection guide. Stage
Parameters
Observation method
Impact
Action
Pre-analytical
Hemolysis
Visual inspection of blood specimen
Establish criteria for specimen rejection (e.g. hemolysis observed = reject)
Pre analytical
Clotting/cell aggregation
Visual inspection of blood specimen
Extreme storage conditions may result in excessive RBC lysis during transport resulting in comprised specimen integrity Inability to accurately characterize immune compartment due to selective loss or alterations in subpopulations
Pre-analytical
Partial draw
Visual inspection of blood specimen
Post-analytical
Light scatter changes
Visual inspection of forward and side scatter plot
Incomplete blood draw may result in increased concentration of anticoagulant or preservative resulting in hypertonic conditions with potentially adverse effects Storage time and temperature may adversely impact cell populations with cytoplasmic granules (e.g. granulocytes), degranulation results in decreased side scatter
specimen stability for flow cytometric assays include the assay format, the choice of anticoagulant or cell stabilization reagent, post-collection specimen processing, temperature, and shipping conditions. The approach suggested within this manuscript is intended to serve as a framework for the evaluation of sample stability as it relates to utilizing a central facility for processing and analysis. As each assay application is unique; it is important to structure the evaluation of stability in a manner that is appropriate for the intended use of the assay. If a given parameter does not meet the stability acceptance criteria, increased stability may be achieved by re-optimizing the assay design (e.g. clone selection, fluorochrome assignment, gating strategy).
Acknowledgments The authors thank the members of the American Association of Pharmaceutical Scientists for review of the manuscript. The assistance of Sarah Livingston in preparing the flow charts is gratefully acknowledged.
References BD Biosciences, 2011. BD multitest™ 6-color TBNK reagent with Trucount tubes. Product Number 23-10834-02. BD Biosciences, San Jose, CA (http:// www.bdbiosciences.com). Beckman Coulter, 2012. CYTO-STAT® tetraCHROME™ CD45-FITC/CD4-RD1/ CD8-ECD/CD3-PC5 and CYTO-STAT® tetraCHROME™ CD45-FITC/CD56RD1/CD19-ECD/CD3-PC5. Product Number 4238068-KE. Beckman Coulter, Brea, CA (http://www.beckmancoulter.com). Belouski, S.S., Wallace, D., Weisman, M., Ishimori, M., Hendricks, L., Zack, D., Vincent, M., Rasmussen, E., Ferbas, J., Chung, J., 2010. Sample stability and variability of B-cell subsets in blood from healthy subjects and patients with systemic lupus erythematosus. Cytometry B Clin. Cytom. 78, 49. Bergeron, M., Nicholson, J.K., Phaneuf, S., Ding, T., Soucy, N., Badley, A.D., Hawley Foss, N.C., Mandy, F., 2002. Selection of lymphocyte gating protocol has an impact on the level of reliability of T-cell subsets in aging specimens. Cytometry 50, 53. Bull, M., Lee, D., Stucky, J., Chiu, Y.L., Rubin, A., Horton, H., McElrath, M.J., 2007. Defining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for HIV vaccine trials. J. Immunol. Methods 322, 57. Carter, P.H., Resto-Ruiz, S., Washington, G.C., Ethridge, S., Palini, A., Vogt, R., Waxdal, M., Fleisher, T., Noguchi, P.D., Marti, G.E., 1992. Flow cytometric
Establish criteria for specimen rejection (e.g. clots are evident = cancelation or clots are evident = proceed with caution and monitor frequency of doublets in light scatter area vs. height plot) Establish criteria for specimen rejection (e.g. bhalf expected volume = reject)
Establish criteria for specimen rejection
analysis of whole blood lysis, three anticoagulants, and five cell preparations. Cytometry 13, 68. Chau, C.H., Rixe, O., McLeod, H., Figg, W.D., 2008. Validation of analytic methods for biomarkers used in drug development. Clin. Cancer Res. 14, 5967. CLSI, 2007. Enumeration of immunologically defined cell populations by flow cytometry; approve guideline H42-A2. Clinical Laboratory Standards Institute vol. 27. Clinical Laboratory Standards Institute. Cunliffe, J., Derbyshire, N., Keeler, S., Coldwell, R., 2009. An approach to the validation of flow cytometry methods. Pharm. Res. 26, 2551. Davis, C., Wu, X., Li, W., Fan, H., Reddy, M., 2011. Stability of immunophenotypic markers in fixed peripheral blood for extended analysis using flow cytometry. J. Immunol. Methods 363, 158. DeSilva, B., Smith, W., Weiner, R., Kelley, M., Smolec, J., Lee, B., Khan, M., Tacey, R., Hill, H., Celniker, A., 2003. Recommendations for the bioanalytical method validation of ligand-binding assays to support pharmacokinetic assessments of macromolecules. Pharm. Res. 20, 1885. Donnenberg, A.D., Donnenberg, V.S., 2007. Rare-event analysis in flow cytometry. Clin. Lab. Med. 27, 627 (viii). Elghetany, M.T., Davis, B.H., 2005. Impact of preanalytical variables on granulocytic surface antigen expression: a review. Cytometry B Clin. Cytom. 65, 1. Fowke, K.R., Behnke, J., Hanson, C., Shea, K., Cosentino, L.M., 2000. Apoptosis: a method for evaluating the cryopreservation of whole blood and peripheral blood mononuclear cells. J. Immunol. Methods 244, 139. Green, C.L., Brown, L., Stewart, J.J., Xu, Y., Litwin, V., Mc Closkey, T.W., 2011. Recommendations for the validation of flow cytometric testing during drug development: I instrumentation. J. Immunol. Methods 363, 104. ISO Guide 30/92-2.7. In: Terms and definitions used in connection with reference materials. Jayachandran, M., Miller, V.M., Heit, J.A., Owen, W.G., 2012. Methodology for isolation, identification and characterization of microvesicles in peripheral blood. J. Immunol. Methods 375, 207. Kuhne, T., Hornstein, A., Semple, J., Chang, W., Blanchette, V., Freedman, J., 1995. Flow cytometric evaluation of platelet activation in blood collected into EDTA vs. Diatube-H, a sodium citrate solution supplemented with theophylline, adenosine, and dipyridamole. Am. J. Hematol. 50, 40–45. Lee, J.W., Devanarayan, V., Barrett, Y.C., Weiner, R., Allinson, J., Fountain, S., Keller, S., Weinryb, I., Green, M., Duan, L., Rogers, J.A., Millham, R., O'Brien, P.J., Sailstad, J., Khan, M., Ray, C., Wagner, J.A., 2006. Fit-for-purpose method development and validation for successful biomarker measurement. Pharm. Res. 23, 312. Maecker, H.T., Hassler, J., Payne, J.K., Summers, A., Comatas, K., Ghanayem, M., Morse, M.A., Clay, T.M., Lyerly, H.K., Bhatia, S., Ghanekar, S.A., Maino, V.C., Delarosa, C., Disis, M.L., 2008. Precision and linearity targets for validation of an IFNgamma ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides. BMC Immunol. 9, 9. Mallone, R., Mannering, S.I., Brooks-Worrell, B.M., Durinovic-Bello, I., Cilio, C.M., Wong, F.S., Schloot, N.C., T-Cell Workshop Committee, I.o.D.S., 2011. Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society. Clin. Exp. Immunol. 163, 33.
8
L. Brown et al. / Journal of Immunological Methods 418 (2015) 1–8
Mandy, F.F., Nicholson, J.K., McDougal, J.S., Cdc, 2003. Guidelines for performing single-platform absolute CD4+ T-cell determinations with CD45 gating for persons infected with human immunodeficiency virus. Centers for Disease Control and Prevention. MMWR. Recommendations and reports: morbidity and mortality weekly report. Recommendations and Reports/Centers for Disease Control 52, p. 1. McCarthy, D.A., 2007. Flow Cytometry: Principles and Applications. In: Macey, M.G. (Ed.), Humana Press Inc, Totowa, NJ. Narayanan, S., 2000. The preanalytic phase. An important component of laboratory medicine. Am. J. Clin. Pathol. 113, 429. Ng, A.A., Lee, B.T., Teo, T.S., Poidinger, M., Connolly, J.E., 2012. Optimal cellular preservation for high dimensional flow cytometric analysis of multicentre trials. J. Immunol. Methods 385, 79. O'Hara, D.M., Xu, Y., Liang, Z., Reddy, M.P., Wu, D.Y., Litwin, V., 2011. Recommendations for the validation of flow cytometric testing during drug development: II assays. J. Immunol. Methods 363, 120. Olson, W.C., Smolkin, M.E., Farris, E.M., Fink, R.J., Czarkowski, A.R., Fink, J.H., Chianese-Bullock, K.A., Slingluff Jr., C.L., 2011. Shipping blood to a central laboratory in multicenter clinical trials: effect of ambient temperature on specimen temperature, and effects of temperature on mononuclear cell yield, viability and immunologic function. J. Transl. Med. 9, 26. Olteanu, H., Schur, B.C., Harrington, A.M., Kroft, S.H., 2012. Time and temperature stability of T-cell subsets evaluated by a dual-platform method. Am. J. Blood Res. 2, 128. Plate, M.M., Louzao, R., Steele, P.M., Greengrass, V., Morris, L.M., Lewis, J., Barnett, D., Warrino, D., Hearps, A.C., Denny, T., Crowe, S.M., 2009. Evaluation of the blood stabilizers TransFix and Cyto-Chex BCT for lowcost CD4 T-cell methodologies. Viral Immunol. 22, 329. Repo, H., Jansson, S.E., Leirisalo-Repo, M., 1995. Anticoagulant selection influences flow cytometric determination of CD11b upregulation in vivo and ex vivo. J. Immunol. Methods 185, 65.
Robert, S., Poncelet, P., Lacroix, R., Arnaud, L., Giraudo, L., Hauchard, A., Sampol, J., Dignat-George, F., 2009. Standardization of platelet-derived microparticle counting using calibrated beads and a Cytomics FC500 routine flow cytometer: a first step towards multicenter studies? J. Thromb. Haemost. (JTH) 7, 190–197. Son, B.K., Roberts, R.L., Ank, B.J., Stiehm, E.R., 1996. Effects of anticoagulant, serum, and temperature on the natural killer activity of human peripheral blood mononuclear cells stored overnight. Clin. Diagn. Lab. Immunol. 3, 260. Stelzer, G.T., Marti, G., Hurley, A., McCoy Jr., P., Lovett, E.J., Schwartz, A., 1997. U.S.–Canadian consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: standardization and validation of laboratory procedures. Cytometry 30, 214. Tanqri, S., Vall, H., Kaplan, D., Hoffman, B., Purvis, N., Porwit, A., Hunsberger, B., Shankey, T.V., on behalf of, I.I.W.G., 2013. Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS – part III – analytical issues. Cytometry B Clin. Cytom. 84, 291. von Pape, K.W., Aland, E., Bohner, J., 2000. Platelet function analysis with PFA-100 in patients medicated with acetylsalicylic acid strongly depends on concentration of sodium citrate used for anticoagulation of blood sample. Thromb. Res. 98, 295. Warrino, D.E., DeGennaro, L.J., Hanson, M., Swindells, S., Pirruccello, S.J., Ryan, W.L., 2005. Stabilization of white blood cells and immunologic markers for extended analysis using flow cytometry. J. Immunol. Methods 305, 107. Wood, B., Jevremovic, D., Béné, M.C., Yan, M., Jacobs, P., Litwin, V., on behalf of, I.I.W.G., 2013. Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS – part V – assay performance criteria. Cytometry B Clin. Cytom. 84, 315.